BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.     

TAP expression provides a general method for improving the recognition of malignant cells in vivo

A major class of tumors lack expression of the transporters associated with antigen processing (TAP). These proteins are essential for delivery of antigenic peptides into the lumen of the endoplasmic reticulum (ER) and subsequent assembly with nascent major histocompatibility complex (MHC) class I, which results in cell surface presentation of the trimeric complex to cytolytic T lymphocytes. Cytolytic T lymphocytes are major effector cells in immunosurveillance against tumors. Here we have tested the hypothesis that TAP downregulation in tumors allows immunosubversion of this effector mechanism, by establishing a model system to examine the role of TAP in vivo in restoring antigen presentation, immune recognition, and effects on malignancy of the TAP-deficient small-cell lung carcinoma, CMT.64. To test the potential of providing exogenous TAP in cancer therapies, we constructed a vaccinia virus (VV) containing the TAP1 gene and examined whether VV-TAP1 could reduce tumors in mice. The results demonstrate that TAP should be considered for inclusion in cancer therapies, as it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic complement of the tumor or the MHC haplotypes of the host.     

Using the TAP component of the antigen-processing machinery as a molecular adjuvant

We hypothesize that over-expression of transporters associated with antigen processing (TAP1 and TAP2), components of the major histocompatibility complex (MHC) class I antigen-processing pathway, enhances antigen-specific cytotoxic activity in response to viral infection. An expression system using recombinant vaccinia virus (VV) was used to over-express human TAP1 and TAP2 (VV-hTAP1,2) in normal mice. Mice coinfected with either vesicular stomatitis virus plus VV-hTAP1,2 or Sendai virus plus VV-hTAP1,2 increased cytotoxic lymphocyte (CTL) activity by at least 4-fold when compared to coinfections with a control vector, VV encoding the plasmid PJS-5. Coinfections with VV-hTAP1,2 increased virus-specific CTL precursors compared to control infections without VV-hTAP1,2. In an animal model of lethal viral challenge after vaccination, VV-hTAP1,2 provided protection against a lethal challenge of VV at doses 100-fold lower than control vector alone. Mechanistically, the total MHC class I antigen surface expression and the cross-presentation mechanism in spleen-derived dendritic cells was augmented by over-expression of TAP. Furthermore, VV-hTAP1,2 increases splenic TAP transport activity and endogenous antigen processing, thus rendering infected targets more susceptible to CTL recognition and subsequent killing. This is the first demonstration that over-expression of a component of the antigen-processing machinery increases endogenous antigen presentation and dendritic cell cross-presentation of exogenous antigens and may provide a novel and general approach for increasing immune responses against pathogens at low doses of vaccine inocula.     

Welcome to the Swamp: Addressing Community Capacity in Ecohealth Research and Intervention

The standpoint from which this article is written is that of development practitioners who work fairly continuously with community transformation processes, and with their peers in many disciplines who are trying to stimulate and support such processes. Drawing on three case examples, the authors put forward four lessons for an ecosystems approach to health development work. First, health and natural resource management professionals, and the technical solutions they create, cannot, by themselves, solve many of the problems communities face. To be effective, solutions have to address a complex set of variables that may be largely invisible to professionals from outside the communities. Creating a map of the human and natural systems within which a particular human health issue arises is often an important first step. Second, another reason why professionals cannot solve complex health challenges on their own is that, in the end, many of the solutions must be implemented by community people from the inside out. Therefore the “map” needs to include human dynamics and community capacity. Third, identifying and assessing the specific capacities that a community needs to address particular health determinants is therefore an important part of health development work. It is critical that community capacity assessment is not undertaken in the abstract, but rather in a way that links capacity assessment with real, ongoing work and through a participatory process that builds understanding and commitment within the community, and identifies clear pathways for future action. Finally, outside professionals working with a community contribute to its capacity to address critical health challenges, not only because of the technical knowledge and skills they bring, but also through the characteristics and attitudes they exhibit. It is therefore important that professionals build their own capacity to role model effective community practice.     

Social behavior of Matschie's tree kangaroos (Dendrolagus matschiei) and its implications for captive management

Social behavior was studied in four (one male and three females) adult, captivebred Matschie's tree kangaroos (Dendrolagus matschiei) over a 124 day span in a large, naturalistic exhibit at the Woodland Park Zoological Gardens, Seattle, WA. A relatively high rate of social interaction occurred (15.2 per hour), over half of which consisted of approaches and nose contacts. The male initiated 54.1% of all social behaviors. Females tended to respond aggressively toward the male (avoid, bite, cuff, or swipe). The male initiated more affiliative behaviors and the females initiated more agonistic behaviors toward both the male and other females. A clear-cut dominance hierarchy could not be determined; reversals (i.e., cases in which the “subordinate” supplanted the “dominant”) occurred 43% of the time overall. Non-aggressive contact behavior consisted primarily of olfactory examination (nose contact). Allogrooming was extremely rare. The high level of agonism and lack of consistent association among individuals suggests that this species may be solitary in the wild. Two joeys were found dead on the floor during the course of the study, and based on previous findings, infanticide or behavioral stress was suspected to be the cause. Successful reproduction did not occur until females were isolated by removing other conspecifics from the enclosure.     

Surrogate Antigen Processing Mediated by TAP-dependent Antigenic Peptide Secretion

MHC class I proteins assemble with peptides in the ER. The peptides are predominantly generated from cytoplasmic proteins, probably by the action of the proteasome, a multicatalytic proteinase complex. Peptides are translocated into the ER by the transporters associated with antigen processing (TAP), and bind to the MHC class I molecules before transport to the cell surface. Here, we use a new functional assay to demonstrate that peptides derived from vesicular stomatitis virus nucleoprotein (VSV-N) antigen are actively secreted from cells. This secretion pathway is dependent on the expression of TAP transporters, but is independent of the MHC genotype of the donor cells. Furthermore, the expression and transport of MHC class I molecules is not required. This novel pathway is sensitive to the protein secretion inhibitors brefeldin A (BFA) and a temperature block at 21°C, and is also inhibited by the metabolic poison, azide, and the protein synthesis inhibitor, emetine. These data support the existence of a novel form of peptide secretion that uses the TAP transporters, as opposed to the ER translocon, to gain access to the secretion pathway. Finally, we suggest that this release of peptides in the vicinity of uninfected cells, which we term surrogate antigen processing, could contribute to various immune and secretory phenomena.Protein secretion has traditionally been thought to involve the action of the translocon located in the membrane of the ER of eukaryotic cells. Proteins are recognized cotranslationally when a signal sequence or a signal–anchor sequence emerges from the ribosome (Walter and Johnson, 1994). These sequences are recognized and bound by the signal recognition particle, and the resulting ribosomal complex then interacts with the signal recognition particle receptor on the ER membrane at the translocon (Andrews and Johnson, 1996). This results in the inclusion of proteins within the secretory pathway. This pathway is by far the best described route of protein secretion in eukaryotic cells. Recently, it has been proposed that some proteins are recognized by a component of the translocon, sec 61, exit the ER, and are transported into the cytoplasm where they are degraded (Wiertz et al., 1996).The translocation into the ER of antigenic peptides for presentation by major histocompatibility complex (MHC)¹ class I molecules is largely independent of the translocon. This form of translocation involves the action of two gene products that are members of the ATP binding cassette family. These genes encode transporters associated with antigen processing 1 and 2 (TAP-1 and -2), and have been implicated in the translocation of peptides from the cytoplasm to the lumen of the ER (Deverson et al., 1990; Bahram et al., 1991; Spies and DeMars, 1991; Spies et al., 1992; Gabathuler et al., 1994). After translocation into the ER, antigenic peptides bind to MHC class I molecules composed of a heavy chain (46-kD) and a light chain (12-kD) called β₂m (Nuchtern et al., 1989; Yewdell and Bennink, 1989; van Bleek and Nathenson, 1990; Matsumura et al., 1992), before transport to the cell surface. The assembly and transport of MHC class I molecules appears to be regulated by a series of chaperones that includes calnexin (Degen and Williams, 1991), calreticulin, and tapasin (Sadasivan et al., 1996).High performance liquid chromatography analysis of peptides eluted from acid-treated whole cells or MHC class I molecules has allowed the identification and characterization of the peptides associated with MHC class I molecules (Falk et al., 1990; Rötzschke et al., 1990; van Bleek and Nathenson, 1990). It is proposed that MHC class I molecules determine the final identity of MHC- restricted peptides and have an instructive role, in addition to a selective role, in peptide selection (Wallny et al., 1992). MHC binding to larger peptides followed by protected proteolytic trimming is a possible mechanism that could account for the observed MHC dependency of cellular peptides (Falk et al., 1990). Peptides unable to bind MHC class I because they are in excess or lack the correct MHC binding motif for the MHC haplotype are thus far undetectable by the techniques commonly used in the field, and are presumed to be short lived and degraded (Falk et al., 1990; Rötzschke et al., 1990). Recent results, however, suggest that peptides not able to bind to a MHC class I molecule intracellularly may be found bound to heat shock proteins (HSPs) such as gp96 (grp94; Arnold et al., 1995). These authors show that cellular antigens are represented by peptides associated with gp96 molecules independently of the MHC class I expressed, confirming earlier results (Udono and Srivastava, 1993, 1994). Gp96 extracted from a specific cell is able to induce cross-priming (Udono and Srivastava, 1993, 1994). Finally, two studies have demonstrated that peptides transported into the lumen of the ER, and do not bind to MHC class I molecules, can be transported out of the ER into the cytoplasm again by a process called “efflux” (Momburg et al., 1994; Schumacher et al., 1994), which may involve the action of the TAP molecules or the sec 61 protein associated with the translocon (Wiertz et al., 1996).We have developed a new bioassay to test the hypothesis that peptides translocated into the ER by the action of the TAP molecules become secreted. Using this assay, we present evidence of an alternative secretion pathway that exists in various mammalian cell types. These observations revise the model of peptide catabolism, and may provide an explanation for various immune and secretion phenomena.     

Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites

The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing "help" to only specific APCs. The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface. In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC. Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells. This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal. It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors.     

Monoclonal Antibodies Combined with Adenovirus-Vectored Interferon Significantly Extend the Treatment Window in Ebola Virus-Infected Guinea Pigs

Monoclonal antibodies (MAbs) are currently a promising treatment strategy against Ebola virus infection. This study combined MAbs with an adenovirus-vectored interferon (DEF201) to evaluate the efficacy in guinea pigs and extend the treatment window obtained with MAbs alone. Initiating the combination therapy at 3 days postinfection (d.p.i.) provided 100% survival, a significant improvement over survival with either treatment alone. The administration of DEF201 within 2 d.p.i. permits later MAb use, with protective efficacy observed up to 8 d.p.i.