A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells

Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ producing CD4 T-cells. By contrast, mucosal exposure to ultraviolet light (UV)-inactivated Ct (UV-Ct) generated regulatory T-cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAP) elicited long-lived protection in conventional and humanized mice. UV-Ct-cSAP targeted immunogenic uterine CD11b+CD103− dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b−CD103+ DCs. Regardless of vaccination route, UV-Ct-cSAP induced systemic memory T-cells, but only mucosal vaccination induced effector T-cells that rapidly seeded uterine mucosa with resident memory T-cells (TRM). Optimal Ct clearance required both TRM seeding and subsequent infection-induced recruitment of circulating memory T-cells. Thus, UV-Ct-cSAP vaccination generated two synergistic memory T-cell subsets with distinct migratory properties

Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl-2 family proteins

BACKGROUND: Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions. METHODS: Stable clones overexpressing maspin are established in the mouse mammary tumor TM40D cells. They are treated with staurosporine, TNF-alpha, and serum starvation. The rates of cell apoptosis are analyzed by TUNEL assay. Inhibitors against caspase 8 and 9 are used in the apoptosis assay. Western blot analysis and ribonuclease protection assay (RPA) are performed to examine the expression of Bcl2 family genes. RESULTS: We report that maspin expressing tumor cells have increased rate of apoptosis when they are treated with staurosporine and serum starvation. The effect is not through extracellular maspin. Maspin-mediated apoptosis is partially blocked by caspase 8 and 9 inhibitors, and is accompanied by changes in the Bcl-2 family proteins. Maspin-expressing tumor cells have a reduced level of anti-apoptotic protein Bcl-2, and an increased level of pro-apoptotic protein Bax. The regulation is not controlled at the transcriptional level but is through selective control of Bcl-2 and Bax protein stability. CONCLUSION: Maspin overexpression modulates tumor cell apoptosis through the regulation of Bcl2 family proteins. Such change results in an increased release of cytochrome c from mitochondria, thus the increased apoptosis in maspin-expressing cells. This evidence strongly suggests that the induction of apoptosis in maspin-overexpressing cells represents a major mechanism by which maspin inhibits breast tumor progression

Discovery and Development of Toll-Like Receptor 4 (TLR4) Antagonists: A New Paradigm for Treating Sepsis and Other Diseases

Abstract. Sepsis remains the most common cause of death in intensive care units in the USA, with a current estimate of at least 750,000 cases per year, and 215,000 deaths annually. Despite extensive research still we do not quite understand the cellular and molecular mechanisms that are involved in triggering and propagation of septic injury. Endotoxin (lipopolysaccharide from Gram-negative bacteria, or LPS) has been implicated as a major cause of this syndrome. Inflammatory shock as a consequence of LPS release remains a serious clinical concern. In humans, inflammatory responses to LPS result in the release of cytokines and other cell mediators from monocytes and macrophages, which can cause fever, shock, organ failure and death. A number of different approaches have been investigated to try to treat and/or prevent the septic shock associated with infections caused by Gram-negative bacteria, including blockage of one or more of the cytokines induced by LPS. Recently several novel amphipathic compounds have been developed as direct LPS antagonists at the LPS receptor, TLR4. This review article will outline the current knowledge on the TLR4-LPS synthesis and discuss the signaling, in vitro pre-clinical and in vivo clinical evaluation of TLR4 antagonists and their potential use in sepsis and a variety of diseases such as atherosclerosis as well as hepatic and renal malfunction. KEY WORDS: drug discovery; LPS; sepsis; toll-like receptor antagonists

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

Background: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phageresistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD 50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophag

The Cytosolic Domain of Fis1 Binds and Reversibly Clusters Lipid Vesicles

Every lipid membrane fission event involves the association of two apposing bilayers, mediated by proteins that can promote membrane curvature, fusion and fission. We tested the hypothesis that Fis1, a tail-anchored protein involved in mitochondrial and peroxisomal fission, promotes changes in membrane structure. We found that the cytosolic domain of Fis1 alone binds lipid vesicles, which is enhanced upon protonation and increasing concentrations of anionic phospholipids. Fluorescence and circular dichroism data indicate that the cytosolic domain undergoes a membrane-induced conformational change that buries two tryptophan side chains upon membrane binding. Light scattering and electron microscopy data show that membrane binding promotes lipid vesicle clustering. Remarkably, this vesicle clustering is reversible and vesicles largely retain their original shape and size. This raises the possibility that the Fis1 cytosolic domain might act in membrane fission by promoting a reversible membrane association, a necessary step in membrane fission

A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells

Genital Chlamydia trachomatis (Ct) infection induces protective immunity that depends on interferon-γ–producing CD4 T cells. By contrast, we report that mucosal exposure to ultraviolet light (UV)–inactivated Ct (UV-Ct) generated regulatory T cells that exacerbated subsequent Ct infection. We show that mucosal immunization with UV-Ct complexed with charge-switching synthetic adjuvant particles (cSAPs) elicited long-lived protection in conventional and humanized mice. UV-Ct–cSAP targeted immunogenic uterine CD11b[superscript +]CD103[superscript –] dendritic cells (DCs), whereas UV-Ct accumulated in tolerogenic CD11b[superscript –]CD103[superscript +] DCs. Regardless of vaccination route, UV-Ct–cSAP induced systemic memory T cells, but only mucosal vaccination induced effector T cells that rapidly seeded uterine mucosa with resident memory T cells (T[subscript RM] cells). Optimal Ct clearance required both T[subscript RM] seeding and subsequent infection-induced recruitment of circulating memory T cells. Thus, UV-Ct–cSAP vaccination generated two synergistic memory T cell subsets with distinct migratory properties.National Institutes of Health (U.S.) (Grant U54-CA119349)National Institutes of Health (U.S.) (Grant U54-CA151884)National Institutes of Health (U.S.) (Grant R37-EB000244)Prostate Cancer FoundationMIT-Portugal ProgramNational Science Foundation (U.S.). Graduate Research Fellowshi