The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, K b -Y84C, in which two a-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound highaffinity peptide. K b -Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (b 2 m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and b 2 m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by b 2 m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that b 2 m association is also tested by the cell surface quality control that leads to MHC-I endocytosis.

Release of fowlpox virus (FWPV) as extracellular enveloped virus (EEV

Bacterial mastitis is accompanied by a drastic increase in milk somatic cell count (SCC), with neutrophils being the predominant cell type found in the infected quarters. Accumulation and activation of neutrophils at the site of infection require local expression of many inflammatory genes encoding adhesion molecules, chemokines and cytokines. Most of the inflammatory genes contain binding sites for the nuclear factor kappaB (NF-kappaB) within their promoter and therefore partly depend on NF-kappaB for their expression. We thus hypothesized that an increase in NF-kappaB activity in the mammary gland could contribute to development of the neutrophilic inflammation that characterizes mastitis. In an attempt to verify this hypothesis, we first assessed milk cells from healthy and acute and chronic mastitis-affected cows for NF-kappaB activity using electrophoretic mobility shift assays. We next studied the relationships between the intensity of NF-kappaB activity in these cells and the degree of udder inflammation. Active NF-kappaB complexes were undetectable in milk cells from healthy cows, whereas high levels of NF-kappaB activity were always found in cells from cows with acute mastitis. In milk cells obtained from chronic mastitis-affected cows, NF-kappaB activity varied from low to high. Finally, the level of NF-kappaB activity measured in milk cells from chronic mastitis-affected cows was not correlated to SCC or to the proportion of neutrophils present in milk samples, but was highly correlated with the expression level of interleukin-8 and granulocyte/macrophage colony-stimulating factor, two NF-kappaB-dependent cytokines crucially involved in initiation and perpetuation of neutrophilic inflammation. These results suggest that NF-kappaB might play a role in mastitis pathogenesis.

Bovine subclinical mastitis can be defined as a moderated inflammatory disease characterized by a persistent accumulation of neutrophils in milk. As GMCSF-mediated delay of neutrophil apoptosis contributes to the accumulation of inflammatory cells at the site of inflammation in many human diseases, we sought to determine whether subclinical mastitis in cows is also associated with a GMCSF-dependent increase in milk-neutrophil survival. We first addressed the hypothesis that GMCSF delays bovine neutrophil apoptosis by activation of the signal transducer and activator of transcription (STAT) family members STAT3 and STAT5, which are critical regulators of the expression of various Bcl-2 family proteins. Granulocyte-macrophage colony-stimulating factor significantly delayed apoptosis of blood neutrophils obtained from healthy cows. In these cells, GMCSF activated STAT5, but not STAT3, and induced an increase in the mRNA of the antiapoptotic Bcl-2 member, Bcl-xL. Granulocyte-macrophage colony-stimulating factor-dependent STAT5 activation and up-regulation of Bcl-xL mRNA were blocked by the Jak inhibitor, AG-490. This inhibition was associated with abrogation of the prosurvival effect of GMCSF, demonstrating a key role for STAT5 in delayed neutrophil apoptosis. We further found that GMCSF expression was increased in milk cells from cows affected with subclinical mastitis. Neutrophils from these cows demonstrated a significant delay of apoptosis as compared with neutrophils obtained from healthy cows and were unresponsive to GMCSF. Active STAT5 complexes were detected in these neutrophils. Finally, in the presence of AG-490, apoptosis was induced and a time-dependent down-regulation of Bcl-xL mRNA was observed in milk neutrophils from mastitis-affected cows. These results indicate that neutrophil survival is enhanced in milk of subclinical mastitis-affected cows and suggest a role for a GMCSF-activated STAT5 signaling pathway in this phenomenon. This pathway could thus represent a target for the control of persistent accumulation of neutrophils in the bovine mammary gland.