DNA vaccination has emerged as a promising strategy for cancer immunotherapy. However, since DNA vaccines have low immunogenicity, various strategies have been developed to enhance the potency of DNA vaccines. In the current study, we aim to determine whether the potency of the DNA vaccine encoding human papillomavirus type 16 (HPV-16) E7 antigen can be enhanced by IL-2. We have generated a DNA vaccine encoding IL-2 linked to HPV-16 E7 antigen. Our results indicate that the DNA vaccine encoding a fusion of IL-2 and E7 proteins generated the highest frequency of E7-specific CD8(+) T cells. We also found that the DNA vaccine encoding a fusion of IL-2 and E7 proteins generated the strongest protective as well as therapeutic anti-tumor effect against E7-expressing tumors. In addition, it was observed that CD8(+) T cells were mainly responsible for the antitumor effect generated by the DNA vaccine encoding a fusion of IL-2 and E7 proteins. Thus, we conclude that the linkage of IL-2 to HPV-16 E7 antigen significantly enhances the DNA vaccine potency against E7-expressing tumors. Our strategy may potentially be used in other antigenic systems to control infectious diseases and/or cancer.

BackgroundEgg white must provide nutrients and protection to the developing avian embryo. One way in which this is achieved is an arsenal of antimicrobial proteins and peptides which are essentially extensions of the innate immune system. Gallin is a recently identified member of a family of peptides that are found in egg white. The function of this peptide family has not been identified and they are potentially antimicrobial.ResultsWe have confirmed that there are at least 3 forms of the gallin gene in the chicken genome in 3 separate lines of chicken, all the forms are expressed in the tubular cells of the magnum region of the oviduct, consistent with its presence in egg white. mRNA expression levels are in the order 10,000 times greater in the magnum than the shell gland. The conservation between the multiple forms of gallin in the chicken genome compared with the conservation between gallin and other avian gallin like peptides, suggests that the gene duplication has occurred relatively recently in the chicken lineage. The gallin peptide family contains a six cysteine motif (C-X5-C-X3-C-X11-C-X3-C-C) found in all defensins, and is most closely related to avian beta-defensins, although the cysteine spacing differs. Further support for the classification comes from the presence of a glycine at position 10 in the 41 amino acid peptide. Recombinant gallin inhibited the growth of Escherischia coli (E. coli) at a concentration of 0.25 μM confirming it as part of the antimicrobial innate immune system in avian species.ConclusionsThe relatively recent evolution of multiple forms of a member of a new defensin related group of peptides that we have termed ovodefensins, may be an adaptation to increase expression or the first steps in divergent evolution of the gene in chickens. The potent antimicrobial activity of the peptide against E. coli increases our understanding of the antimicrobial strategies of the avian innate immune system particularly those of the egg white and the evolution of the defensin family. The potential of this peptide and others in the family can now be investigated in a number of novel antimicrobial roles.

Natural antimicrobial peptides are present in different compartments (eggshell, egg white, and vitelline membranes) of the hen egg and are expected to be involved in the protection of the embryo during its development and to contribute to the production of pathogen-free eggs. In the present study, we used vitelline membranes from hen (Gallus gallus) eggs as a source of avian ␤-defensin 11 (AvBD11). A purification scheme using affinity chromatography and reverse-phase chromatography was developed. Purified AvBD11 was analyzed by a combination of mass spectrometry approaches to characterize its primary sequence and structure. A monoisotopic molecular species at [M ؉ H]؉ of 9,271.56 Da was obtained, and its N-and C-terminal sequences were determined. We also examined posttranslational modifications and identified the presence of 6 internal disulfide bonds. AvBD11 was found to exhibit antimicrobial activity toward both Gram-positive and Gram-negative bacteria.The avian egg is a unique and original biological system which is formed according to a well-defined spatial and temporal sequence as it passes along the reproductive tract of the hen. The segments of the oviduct involved in the egg formation are the infundibulum, the magnum, the isthmus, and the uterus. Each segment expresses and secretes specific molecules that become successively incorporated into the vitelline membranes in the infundibulum, the egg white in the magnum, the eggshell membranes in the isthmus, and the eggshell in the uterus. Thus, the hen deposits into the egg, which can be considered a closed chamber, all the nutrients and protective systems that are necessary to support the development of an embryo during 21 days of incubation. Recently, transcriptomic and proteomic approaches have identified almost 1,000 putative proteins and peptides in the various compartments of eggs (6). Among these are a number of polypeptides that are likely to resist microbial contamination of the eggs, some of which belong to the family of avian ␤-defensins (AvBDs).A total of 14 avian ␤-defensin genes have been identified through in silico studies. Because most of them were simultaneously described by two research groups, some confusion was generated due to different nomenclatures (9,10,18,31). In a collaborative venture, we proposed a novel nomenclature that adopted the numbering system used by Xiao et al. (31) and replaced the term "gallinacin," used formerly by Lehrer's group, with "avian ␤-defensin" (AvBD) (19). AvBDs are small cationic nonglycosylated peptides (1 to 9 kDa). Some of them were successfully tested for their antimicrobial activity, including chicken AvBDs AvBD1, -2, -7, and -9, turkey AvBD1 and -2, ostrich AvBDs AvBD1, -2, -7, and -8, and penguin AvBD103b (3, 30). In mammals, ␤-defensins are molecules of innate and adaptive immunity, with a broad spectrum of antimicrobial activity (1). ␤-Defensin molecules possess six highly conserved cysteines with the following consensus sequence motif, where C is a cysteine, G a glycine, and X any amino acid: X...

Background: Ovalbumin-related protein X (OVAX) is an uncharacterized ovalbumin-serpin.Results: This egg white-specific serpin lacks protease inhibitory activity, but unlike its ovalbumin homolog, OVAX exhibits antibacterial properties, partly through its heparin-binding site(s).Conclusion: OVAX, a non-inhibitory serpin is a heparin-binding molecule with antibacterial activity.Significance: OVAX participates in egg defense and constitutes a natural agent against Listeria and Salmonella.

Background information. Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage).Results. Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high-and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3-or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. Conclusions.Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.

No abstract

Silicosis is an occupational pneumoconiosis caused by inhalation of crystalline silica. It leads to the formation of fibrohyalin nodes that result in progressive fibrosis. Alternatively, emphysema may occur, with abnormal destruction of collagen fibres in the advanced stages. Although the pathophysiological mechanisms remain unclear, it has been established that the lung responds to silica by massive enrollment of alveolar macrophages, triggering an inflammatory cascade of reactions. An imbalance in the expression of lung proteases and their inhibitors is implicated in extracellular matrix remodelling and basement membrane disruption. Moreover, exposure to silica can initiate apoptotic cell death of macrophages. This review summarises the current knowledge on cysteine cathepsins that have been ignored so far during silicosis and outlines the recent progress on cellular pathways leading to silica-induced caspase activation, which have been partly delineated.

No abstract