ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L ؊/؊ and ISG15 ؊/؊ mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.One of the earliest host responses to viral infection is the production of type I interferons (IFN-␣ and -␤) and the subsequent upregulation of IFN-stimulated genes (ISGs). These ISGs generate an antiviral state in nearby cells and also play an important role in shaping the host innate and adaptive immune response (26, 28). We and others have recently identified ISG15 as a critical IFN-induced antiviral molecule. Overexpression of ISG15 by a recombinant Sindbis virus protected IFN-␣␤ receptor-deficient mice from lethality (13). In a cell culture system, the overexpression of ISG15 also inhibited the release of human immunodeficiency virus virions (20) and decreased alphavirus replication (32). Finally, mice lacking ISG15 are susceptible to several human pathogens, including influenza A and B viruses, herpesviruses, and Sindbis viruses (14). Though it is clear that ISG15 functions as an antiviral molecule, its mechanism and site of action remain poorly understood.ISG15, a 17-kDa ubiquitin-like molecule, contains two ubiquitin domains, including a carboxy-terminal LRLRGG motif, through which it forms conjugates with target proteins (15,19). ISG15 conjugation of target proteins (ISGylation) utilizes an IFN-induced conjugation cascade which includes an E1 (UbE1L/Uba7), an E2 (UbcH8), several E3 ligases (EFP, HHARI, and Herc5), and a deconjugating protease (UBP43/ USP18) (1,11,17,22,31,33,35). Activation of this pathway results in the conjugation of ISG15 to over 100 known target proteins that encompass multiple biological pathways (8,16,30,34). In addition to forming conjugates, free ISG15 also accumulates within cells and is released into the sera of patients following stimulation with IFN (5). Recombinant ISG15 has been reported to function as a cytokine, stimulating IFN-␥ production, NK cell proliferation, neutrophil chemotaxis, and dendritic cell maturation (4,(23)(24)(25). During viral infection in mice, ISG15 exists in three forms: (i) unconjugated within cells, (ii) conjugated to target proteins, and (iii) released into the serum (14). Studies with Sindbis virus suggest that the conjugated form of ISG15 mediates its antiviral activity. The increased lethality seen in ISG15 Ϫ/Ϫ mice can be rescued by a recombinant virus expressing wild-type ISG15 but not mutant ISG15, which cannot form conjugates in vitro (14). In contrast, two recent...

The vitreous humor of the eye is a biological hydrogel principally composed of collagen fibers interspersed with hyaluronic acid. Certain pathological conditions necessitate its removal and replacement. Current substitutes, like silicone oils and perfluorocarbons, are not biomimetic and have known complications. In this study, we have developed an in situ forming two-component biomimetic hydrogel with tunable mechanical and osmotic properties. The components are gellan, an analogue of collagen, and poly(methacrylamide-co-methacrylate), an analogue of hyaluronic acid; both endowed with thiol side groups. We used response surface methodology to consider seventeen possible hydrogels to determine how each component affects the optical, mechanical, sol-gel transition temperature and swelling properties. The optical and physical properties of the hydrogels were similar to vitreous. The shear storage moduli ranged from 3 to 358 Pa at 1Hz and sol-gel transition temperatures from 35.5 to 43 °C. The hydrogel had the ability to remain swollen without degradation for four weeks in vitro. Three hydrogels were tested for biocompatibility on primary porcine retinal pigment epithelial cells, human retinal pigment epithelial cells, and fibroblast (3T3/NIH) cells, by electric cell-substrate impedance sensing system. The two-component hydrogels allowed for the tuning and optimizing of mechanical, swelling, and transition temperature to obtain three biocompatible hydrogels with properties similar to vitreous. Future studies include testing of the optimized hydrogels in animal models for use as a long-term substitute, whose preliminary results are mentioned.

The natural vitreous is a biological hydrogel consisting primarily of a collagen and anionic hyaluronate. It is surgically removed in many ocular diseases and replaced with fluids, gases, or silicone oils. We have been interested in developing synthetic hydrogels as vitreous substitutes. In this study, we combined the stiffness and hydrophobicity of polymethacrylamide (PMAM) and the anionic nature of poly-methacrylate (PMAA) to make copolymers that would mimic the natural vitreous. We used bis-methacryloyl cystamine (BMAC) to introduce thiol groups for reversible crosslink. The Mn of copolymers ranged from ~100 k to ~200 k Da (polydis-peristy index of 1.47–2.63) and their composition as determined by titration, 1H NMR and disulfide test were close to the feed ratio. The reactivities of monomers were as follows: MAM > MAA ~ BMAC. Copolymers with higher MAA contents gelled faster, swelled more, and had higher storage modulus (1.5 to 100 Pa) comparable to that of the natural vitreous. We evaluated the biocompatibility of copolymers by electric cell-substrate impedance sensing (ECIS) using human retinal pigment epithelial cells, primary porcine retinal pigmented epithelial cells, human microvascular endothelial cells adult dermis, and a fibroblast line 3T3. The biocompatibility decreases as the content of BMAC increases.

ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15؊/؊ mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15 ؊/؊ mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugationdependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo. IMPORTANCE It has previously been demonstrated that ISG15؊/؊ mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15 ؊/؊ mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo. During acute infection, the survival of an organism is dependent on both its ability to inhibit pathogen replication, thus reducing pathogen burden, and its ability to tolerate the ensuing tissue damage incurred both from the pathogen itself, as well as from the immune response mounted against the pathogen (1, 2). Limiting pathogen replication and eventual pathogen clearance is dependent on both the innate and the adaptive immune responses. Of the many genes induced after viral infection, type I interferons ...