The oxazolidinone linezolid is a new antibacterial approved for marketing in 2000 that inhibits bacterial protein synthesis (5,20,41). It represents a new structural class of antibiotics, with activity against several gram-positive organisms, including several resistant strains. Linezolid has been shown to be effective in treating nosocomial pneumonia caused by methicillinsusceptible and -resistant Staphylococcus aureus or multidrugresistant Streptococcus pneumonia and skin and soft tissue infections caused by methicillin-susceptible and -resistant Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus agalactiae. It is also effective against community-acquired pneumonia caused by methicillin-susceptible S. aureus, multidrug-resistant S. pneumoniae, and vancomycin-resistant Enterococcus faecium infections (15, 31).The oxazolidinones inhibit bacterial protein synthesis, although the exact details concerning the mechanism(s) of inhibition are still emerging. Early results demonstrated that the oxazolidinone eperezolid binds to 50S but not 30S ribosomal subunits. Furthermore, binding was inhibited by chloramphenicol and lincomycin (27). Cross-linking studies have been carried out to identify the sites of oxazolidinone binding. Using ribosomes from Escherichia coli, a number of nucleotide residues in domain V of 23S rRNA were identified, as well as residue A864 of 16S rRNA (28). Colca et al. carried out crosslinking experiments using intact Staphylococcus aureus and showed that tRNA, two ribosomal proteins, and nucleotide A2602 of 23S rRNA all were labeled by the cross-linker (10).The results from mapping oxazolidinone resistance mutations agree with the cross-linking studies. Linezolid-resistant mutants of Halobacterium halobium were isolated and shown to contain single point mutations in the central loop of domain V of 23S rRNA (24). Likewise, Escherichia coli oxazolidinoneresistant mutants contained G2032A and G2447A mutations, which also are in domain V (4, 47

The classical nuclear factor κB (NF-κB) signaling pathway is under the control of the IκB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-κB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-κB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of κBα (IκBα). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Faslpr/lpr), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in urine are significantly increased in DN patients and correlated with proteinuria and MCP-1 levels. Thus our data not only confirm the up-regulation of Fn14/TWEAK pathway in kidney diseases, but also suggest a novel mechanism for its regulation by the generation of sFn14. The correlation of sFn14 levels and disease severity suggest that sFn14 may serve as a potential biomarker for both acute and chronic kidney diseases.