Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge : A proof-of-concept study

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Oral immunization with a novel lipid-based adjuvant protects against genital Chlamydia infection

Oral immunization is attractive as a delivery route because it is needle-free and useful for rapid mass vaccination programs to target pandemics or bioterrorism. This potential has not been realized for human vaccination, due to the requirement of large antigen doses and toxic (to humans) adjuvants to overcome the induction of oral tolerance and potential degradation of antigens in the stomach. To date, only oral vaccines based on live attenuated organisms have been approved for human use. In this study we describe the use of a lipid-based delivery system/adjuvant, Lipid C, for oral immunization to protect mice against genital tract chlamydial infection. Lipid C is formulated from food-grade purified and fractionated triglycerides. Bacterial shedding following vaginal challenge with Chlamydia muridarum was reduced by 50% in female mice orally immunized with the chlamydial major outer membrane protein (MOMP) formulated in Lipid C, protection equivalent to that seen in animals immunized with MOMP admixed with both cholera toxin (CT) and CpG oligodeoxynucleotides (CpG-ODN). Protection was further enhanced when MOMP, CT and CpG were all combined in the Lipid C matrix. Protection correlated with production of gamma interferon (IFN) by splenic T cells, a serum MOMP-specific IgG response and low but detectable levels of MOMP-specific IgA in vaginal lavage.

Transcutaneous immunization with a novel lipid-based adjuvant protects against Chlamydia genital and respiratory infections

Mucosal adjuvants are important to overcome the state of immune tolerance normally associated with mucosal delivery and to enhance adaptive immunity to often-weakly immunogenic subunit vaccine antigens. Unfortunately, adverse side effects of many experimental adjuvants limit the number of adjuvants approved for vaccination. Lipid C is a novel, non-toxic, lipid oral vaccine-delivery formulation, developed originally for oral delivery of the live Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine. In the present study, murine models of chlamydial respiratory and genital tract infections were used to determine whether transcutaneous immunization (TCI) with Lipid C-incorporated protein antigens could elicit protective immunity at the genital and respiratory mucosae. BALB/c mice were immunized transcutaneously with Lipid C containing the chlamydial major outer membrane protein (MOMP), with and without addition of cholera toxin and CpG-ODN 1826 (CT/CpG). Both vaccine combinations induced mixed cell-mediated and mucosal antibody immune responses. Immunization with Lipid C-incorporated MOMP (Lipid C/MOMP), either alone or with CT/CpG resulted in partial protection following live challenge with Chlamydia muridarum as evidenced by a significant reduction in recoverable Chlamydia from both the genital secretions and lung tissue. Protection induced by immunization with Lipid C/MOMP alone was not further enhanced by the addition of CT/CpG. These results highlight the potential of Lipid C as a novel mucosal adjuvant capable of targeting multiple mucosal surfaces following TCI. Protection at both the respiratory and genital mucosae was achieved without the requirement for potentially toxic adjuvants, suggesting that Lipid C may provide a safe effective mucosal adjuvant for human vaccination.

Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection

Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of the C. trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

Immunization with a MOMP-based vaccine protects mice against a Pulmonary Chlamydia challenge and identifies a disconnection between infection and pathology

Chlamydia pneumoniae is responsible for up to 20% of community acquired pneumonia and can exacerbate chronic inflammatory diseases. As the majority of infections are either mild or asymptomatic, a vaccine is recognized to have the greatest potential to reduce infection and disease prevalence. Using the C. muridarum mouse model of infection, we immunized animals via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, with recombinant chlamydial major outer membrane protein (MOMP) combined with adjuvants CTA1-DD or a combination of cholera toxin/CpG-oligodeoxynucleotide (CT/CpG). Vaccinated animals were challenged IN with C. muridarum and protection against infection and pathology was assessed. SL and TC immunization with MOMP and CT/CpG was the most protective, significantly reducing chlamydial burden in the lungs and preventing weight loss, which was similar to the protection induced by a previous live infection. Unlike a previous infection however, these vaccinations also provided almost complete protection against fibrotic scarring in the lungs. Protection against infection was associated with antigen-specific production of IFNγ, TNFα and IL-17 by splenocytes, however, protection against both infection and pathology required the induction of a similar pro-inflammatory response in the respiratory tract draining lymph nodes. Interestingly, we also identified two contrasting vaccinations capable of preventing infection or pathology individually. Animals IN immunized with MOMP and either adjuvant were protected from infection, but not the pathology. Conversely, animals TC immunized with MOMP and CTA1-DD were protected from pathology, even though the chlamydial burden in this group was equivalent to the unimmunized controls. This suggests that the development of pathology following an IN infection of vaccinated animals was independent of bacterial load and may have been driven instead by the adaptive immune response generated following immunization. This identifies a disconnection between the control of infection and the development of pathology, which may influence the design of future vaccines.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

Purpose The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

Instant coffee with high chlorogenic acid levels protects humans against oxidative damage of macromolecules

Scope: Coffee is among the most frequently consumed beverages. Its consumption is inversely associated to the incidence of diseases related to reactive oxygen species; the phenomenon may be due to its antioxidant properties. Our primary objective was to investigate the impact of consumption of a coffee containing high levels of chlorogenic acids on the oxidation of proteins, DNA and membrane lipids; additionally, other redox biomarkers were monitored in an intervention trial. Methods and results: The treatment group (n=36) consumed instant coffee co-extracted from green and roasted beans, whereas the control consumed water (800 mL/P/day, 5 days). A global statistical analysis of four main biomarkers selected as primary outcomes showed that the overall changes are significant. 8-Isoprostaglandin F2α in urine declined by 15.3%, 3-nitrotyrosine was decreased by 16.1%, DNA migration due to oxidized purines and pyrimidines was (not significantly) reduced in lymphocytes by 12.5 and 14.1%. Other markers such as the total antioxidant capacity were moderately increased; e.g. LDL and malondialdehyde were shifted towards a non-significant reduction. Conclusion: The oxidation of DNA, lipids and proteins associated with the incidence of various diseases and the protection against their oxidative damage may be indicative for beneficial health effects of coffee.

Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins

Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

The Trans-Pacific partnership protects old companies at the expense of the new

Under this trade agreement, intellectual property will be transformed into a means of protecting the investments of multinational companies in culture, advertising, and medicine, writes Matthew Rimmer. The Trans-Pacific Partnership (TPP) is a blockbuster trade deal, which will transform the intellectual property of the Pacific Rim. The Australian Prime Minister Malcolm Turnbull has argued the trade agreement is a "gigantic foundation stone" for the country's future prosperity. Nonetheless, though the final text remains unreleased, it's clear from what we do know that the Intellectual Property Chapter is set to protect legacy intellectual property industries - rather than support disruptive technologies and dynamic innovation.

Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case–control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of ‘pore’ function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln–270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory ‘pore’ function.

Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.

Glutathione peroxidase3 of Saccharomyces cerevisiae protects phospholipids during cadmium-induced oxidative stress

The present study was undertaken to determine the role of glutathione peroxidase3 (gpx3) in phospholipid protection in cells. Wild-type (WT) cells showed an overall increase in phospholipids upon 50 mu M cadmium (Cd)-treatment, whereas an untreated gpx3 Delta strain showed a drastic reduction in overall phospholipids which was further reduced with 50 mu M Cd. In WT cells, Cd-exposure increased the short chain fatty acids and decreased the unsaturated fatty acids and the magnitude was high in Cd-treated gpx3 Delta cells. Purified recombinant gpx3p showed higher activity with phospholipid hydroperoxides than shorter hydroperoxides. An increase in gpx activity was observed in Cd-treated WT cells and no such alteration was observed in gpx3 Delta. WT cells treated with Cd showed an increase in MDA over untreated, while untreated gpx3 Delta cells themselves showed a higher level of MDA which was further enhanced with Cd-treatment. Iron, zinc and calcium levels were significantly altered in WT and gpx3 Delta cells during Cd-treatment.