The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5−/−mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.

Alternatively polarized macrophages (MU) shape the microenvironment of hepatocellular carcinoma (HCC) and temper anticancer immune responses. We investigated if sorafenib alters the HCC microenvironment by restoring classical macrophage polarization and triggering tumor-directed natural killer (NK) cell responses. In vivo experiments were conducted with sorafenib (25 mg/kg)-treated C57BL/6 wildtype as well as hepatitis B virus (HBV) and lymphotoxin transgenic mice with and without HCC. Monocyte-derived MU or tumor-associated macrophages (TAM) isolated from HCC tissue were treated with sorafenib (0.07-5.0 lg/mL) and cocultured with autologous NK cells. MU and NK cell activation was analyzed by flow cytometry and killing assays, respectively. Cytokine and growth factor release was measured by enzyme-linked immunosorbent assay. Short-term administration of sorafenib triggered activation of hepatic NK cells in wildtype and tumor-bearing mice. In vitro, sorafenib sensitized MU to lipopolysaccharide, reverted alternative MU polarization and enhanced IL12 secretion (P 5 0.0133). NK cells activated by sorafenib-treated MU showed increased degranulation (15.3 6 0.2% versus 32.0 6 0.9%, P < 0.0001) and interferon-gamma (IFN-c) secretion (2.1 6 0.2% versus 8.0 6 0.2%, P < 0.0001) upon target cell contact. Sorafenib-triggered NK cell activation was verified by coculture experiments using TAM. Sorafenib-treated MU increased cytolytic NK cell function against K562, Raji, and HepG2 target cells in a dose-dependent manner. Neutralization of interleukin (IL)12 or IL18 as well as inhibition of the nuclear factor kappa B (NF-jB) pathway reversed NK cell activation in MU/NK cocultures. Conclusion: Sorafenib triggers proinflammatory activity of TAM and subsequently induces antitumor NK cell responses in a cytokine-and NF-jB-dependent fashion. This observation is relevant for HCC therapy, as sorafenib is a compound in clinical use that reverts alternative polarization of TAM in HCC. (HEPATOLOGY 2013;57:2358-2368 T umor-associated macrophages (TAM) located in the hepatocellular carcinoma (HCC) environment increase HCC recurrence after resection and reduce patient survival. 1,2 TAM thereby fosters tumor cell proliferation and tumor spread. 3 Natural killer (NK) cell numbers and activity, on the other hand, are associated with lower HCC stages and improved patient survival. 4,5 An outstanding feature of Abbreviations: AFP, alpha-fetoprotein; C57BL/6 wt , C57BL/6

Uveitis is a sight-threatening eye disease in equids known worldwide that leads to considerable pain and suffering. By far the most common type of uveitis in Germany and neighboring countries is classical equine recurrent uveitis (ERU), which is caused by chronic intraocular leptospiral infection and is the main cause of infectious uveitis in horses. Other infectious causes are extremely rare and are usually clinically distinguishable from ERU. ERU can be treated very effectively by vitreous cavity lavage (vitrectomy). For proper indications of this demanding surgery, it is necessary to differentiate ERU from other types of uveitis in which vitrectomy is not helpful. This can be conducted on the basis of anamnesis in combination with ophthalmologic findings and by aqueous humor examination. During vitrectomy, vitreous material is obtained. These vitreous samples have historically been used for numerous etiologic studies. In this way, a chronic intraocular leptospiral infection has been shown to be the cause of typical ERU and, among other findings, ERU has also been recognized as a biofilm infection, providing new insights into the pathogenesis of ERU and explaining some thus far unexplainable phenomena of ERU. ERU may not only have transmissible aspects to some types of uveitis in humans, but may also serve as a model for a spontaneously occurring biofilm infection. Vitreous material obtained during therapeutically indicated vitrectomy can be used for further studies on in vivo biofilm formation, biofilm composition and possible therapeutic approaches.

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Background:It is important to understand whether proteasomal dysfunction and endoplasmic reticulum (ER) stress can influence prion propagation. Results: Both events lead to an increase of PrP aggregates in the secretory pathway and increased pathologic prion protein in infected cells. Conclusion: Our data suggest a novel pathway that contributes to prion propagation. Significance: These findings might be of relevance for the pathogenesis of sporadic prion diseases.