HIV-specific CD8+ T cells exhibit markedly reduced levels of Bcl-2 and Bcl-xL

Human immunodeficiency virus-specific CD8(+) T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-x(L) in HIV-specific CD8(+) T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8(+) T cells compared with CMV-specific and total CD8(+) T cells from HIV-infected individuals as well as total CD8(+) T cells from healthy donors. CD8(+) T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8(+) T cells from HIV-infected individuals. HIV-specific CD8(+) T cells also had significantly lower levels of Bcl-x(L) compared with CMV-specific CD8(+) T cells. Finally, IL-15 induces both Bcl-2 and Bcl-x(L) expression in HIV-specific and total CD8(+) T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-x(L) may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8(+) T cells and suggest a possible mechanism by which IL-15 increases their survival.     

IFN-alpha priming results in a gain of proinflammatory function by IL-10: implications for systemic lupus erythematosus pathogenesis

Interleukin-10 is a predominantly anti-inflammatory cytokine that inhibits macrophage and dendritic cell function, but can acquire proinflammatory activity during immune responses. We investigated whether type I IFNs, which are elevated during infections and in autoimmune diseases, modulate the activity of IL-10. Priming of primary human macrophages with low concentrations of IFN-alpha diminished the ability of IL-10 to suppress TNF-alpha production. IFN-alpha conferred a proinflammatory gain of function on IL-10, leading to IL-10 activation of expression of IFN-gamma-inducible, STAT1-dependent genes such as IFN regulatory factor 1, IFN-gamma-inducible protein-10 (CXCL10), and monokine induced by IFN-gamma (CXCL9). IFN-alpha priming resulted in greatly enhanced STAT1 activation in response to IL-10, and STAT1 was required for IL-10 activation of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma expression in IFN-alpha-primed cells. In control, unprimed cells, IL-10 activation of STAT1 was suppressed by constitutive activity of protein kinase C and Src homology 2 domain-containing phosphatase 1. These results demonstrate that type I IFNs regulate the balance between IL-10 anti- and proinflammatory activity, and provide insight into molecular mechanisms that regulate IL-10 function. Gain of IL-10 proinflammatory functions may contribute to its pathogenic role in autoimmune diseases characterized by elevated type I IFN levels, such as systemic lupus erythematosus.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.     

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the ⁸F1⁹F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to ⁸F1⁹F1 and that UR binding to ⁸F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.     

Multi-factorial modulation of IGD motogenic potential in MSF (Migration Stimulating Factor)

Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as ³FnI, ⁵FnI, ⁷FnI and ⁹FnI, respectively. We have previously reported that mutation of IGD motifs in modules ⁷FnI and ⁹FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in ³FnI and ⁵FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within ^1-5FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in ⁷FnI and ⁹FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.     

Follicular atresia during Dacus oleae oogenesis

Programmed cell death, constitutes a common fundamental incident that occurs during oogenesis in a variety of different animals. It plays a significant role in the maturation process of the female gamete and also in the removal of abnormal and superfluous cells at certain checkpoints of development. In the present study, we demonstrate the existence of follicular atresia during mid-oogenesis in the olive fruit fly Dacus oleae (Tephritidae). The number of atretic follicles increases following the age of the fly, suggesting for the presence of an age-susceptible process. The atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton alterations, as revealed by propidium iodide staining, TUNEL labeling and phalloidin-FITC staining. Conventional light and electron microscopy disclose that the nurse cell remnants are phagocytosed by the adjacent follicle cells. The follicular epithelium also eliminates the oocyte through phagocytosis, resulting to an egg chamber with no compartmentalized organization. The data presented herein are very similar compared to previous reported results in other Diptera species, strongly suggesting the occurrence of a phylogenetically conserved mechanism of follicular atresia. All these observations also support the notion that mid-oogenesis in D. oleae may be the critical regulation point at which superfluous and defective egg chambers are selectively eliminated before they reach maturity.