Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine ...production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.
In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.
The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.
Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.
The rhoptry of the malaria parasite Plasmodium falciparum is an unusual secretory organelle that is thought to be related to secretory lysosomes in higher eukaryotes. Rhoptries contain an extensive ...collection of proteins that participate in host cell invasion and in the formation of the parasitophorous vacuole, but little is known about sorting signals required for rhoptry protein targeting. Using green fluorescent protein chimeras and in vitro pull-down assays, we performed an analysis of the signals required for trafficking of the rhoptry protein RAP1. We provide evidence that RAP1 is escorted to the rhoptry via an interaction with the glycosylphosphatidyl inositol-anchored rhoptry protein RAMA. Once within the rhoptry, RAP1 contains distinct signals for localisation within a sub-compartment of the organelle and subsequent transfer to the parasitophorous vacuole after invasion. This is the first detailed description of rhoptry trafficking signals in Plasmodium.
Mucosal-associated invariant T (MAIT) cells are novel innate-like T cells constituting a significant proportion of circulating and hepatic T cells. Herein, we extensively examine the phenotypical and ...functional alterations of MAIT cells and their regulation in a cohort of 56 patients with Primary Biliary Cholangitis (PBC) and 53 healthy controls (HC). Additionally alterations of MAIT cells were assessed before and after UDCA treatment. Finally the localization of MAIT cell in liver was examined using specific tetramer staining and the underlying mechanisms of these alterations in PBC were explored. Our data demonstrated that the frequency and number of circulating MAIT cells were decreased, whereas hepatic MAIT cells were increased in PBC compared to HC. Moreover, circulating MAIT cells were more activated in PBC than HC, reflected by elevated expression levels of granzyme B. Six months of UDCA treatment significantly attenuated the circulating MAIT cells differences in PBC. Of note, the expression levels of IL-7 were significantly increased in both plasma and liver from PBC as compared to HC, which promoted the production of inflammatory cytokines and granzyme B by inducing signal transduction and activation of transcription 5 (STAT5) phosphorylation in MAIT cells. Finally, cholic acid, one of the major bile acids in liver, upregulated IL-7 expression in hepatocyte cell line L02 by inducing Farnesoid X Receptor (FXR) binding to the IL-7 promoter. Hence MAIT cells are activated and enriched in the liver of PBC. Cholic acid-induced IL-7 production in hepatocytes plays a critical role in regulating MAIT cell function, highlighting that hepatocytes may bridge cholangiocyte injury and innate immunity through a bile acid signaling pathway.
•An indispensable role of MAIT cells in PBC was suggested.•MAIT cells are more activated with increased cytokine production in PBC.•IL-7 enhanced cytokine production in MAIT cells in PBC.•Bile acids can affect MAIT cell functions through regulating expression of IL-7.
Background Soil-transmitted helminths (STH), Schistosoma spp. and Plasmodium falciparum are parasites of major public health importance and co-endemic in many sub-Saharan African countries. ...Management of these infections requires detection and treatment of infected people and evaluation of large-scale measures implemented. Diagnostic tools are available but their low sensitivity, especially for low intensity helminth infections, leaves room for improvement. Antibody serology could be a useful approach thanks to its potential to detect both current infection and past exposure. Methodology We evaluated total IgE responses and specific-IgG levels to 9 antigens from STH, 2 from Schistosoma spp., and 16 from P. falciparum, as potential markers of current infection in a population of children and adults from Southern Mozambique (N = 715). Antibody responses were measured by quantitative suspension array Luminex technology and their performance was evaluated by ROC curve analysis using microscopic and molecular detection of infections as reference. Principal findings IgG against the combination of EXP1, AMA1 and MSP2 (P. falciparum) in children and NIE (Strongyloides stercoralis) in adults and children had the highest accuracies (AUC = 0.942 and AUC = 0.872, respectively) as markers of current infection. IgG against the combination of MEA and Sm25 (Schistosoma spp.) were also reliable markers of current infection (AUC = 0.779). In addition, IgG seropositivity against 20 out of the 27 antigens in the panel differentiated the seropositive endemic population from the non-endemic population, suggesting a possible role as markers of exposure although sensitivity could not be assessed. Conclusions We provided evidence for the utility of antibody serology to detect current infection with parasites causing tropical diseases in endemic populations. In addition, most of the markers have potential good specificity as markers of exposure. We also showed the feasibility of measuring antibody serology with a platform that allows the integration of control and elimination programs for different pathogens.
The cross‐talk of cluster of differentiation (CD)40/CD40 ligand (CD40L) plays a key role in CD4+ T‐cell priming, B‐cell terminal maturation, and immunoglobulin (Ig) class‐switch recombination. ...Genetic defects in the CD40L lead to a disorder characterized by elevated concentrations of serum IgM and immunodeficiency. Patients with primary biliary cirrhosis (PBC) characteristically show circulating antimitochondrial antibodies (AMAs), liver‐infiltrating autoreactive T lymphocytes against mitochondrial antigens, and high levels of IgM. We hypothesized that CD40L may play a key role in the pathogenesis of the elevated serum IgM and analyzed genetic and epigenetic modifications of the gene coding for CD40L in CD4+ and CD8+ T cells isolated from circulating mononuclear cells from PBC patients and healthy controls. We herein demonstrate significantly lower levels of DNA methylation of the CD40L promoter in CD4+ T cells from PBC patients, as compared with controls, and this decreased methylation was inversely correlated with levels of serum IgM in PBC patients.Conclusion: The findings of an absence of genetic modifications of the CD40L gene, in concert with decreased DNA methylation of the CD40L promoter in PBC patients, suggests that environmental factors, rather than genetics, must play a major role in the pathogenesis of elevated serum IgM in PBC. (HEPATOLOGY 2012)
The antimitochondrial response in primary biliary cirrhosis (PBC) is the most highly directed and specific self‐reacting antibody in human immunopathology. Originally, antimitochondrial antibodies ...(AMAs) were detected by indirect immunofluorescence (IIF) and found in approximately 90% of well‐documented patients with PBC. The introduction of recombinant autoantigens and the use of immunoblotting have increased the sensitivity and specificity of AMAs, and they are now considered positive in approximately 95% of patients with PBC. Clearly, accurate autoantibody detection represents one of the fundamental requirements for reliable diagnostics in autoimmunity. To address the 5% of AMA‐negative patients with PBC, we have generated and validated a bead assay for the detection of AMA. We enrolled 120 patients with PBC, including a non‐random group of 30 rigorously proven AMA‐negative patients, 50 healthy subjects, and 74 controls with autoimmune diseases (18 with primary sclerosing cholangitis, 16 with autoimmune hepatitis, and 40 with systemic lupus erythematosus). Individual bead assays were done with the three mitochondrial autoantigens, PDC‐E2, BCOADC‐E2, and OGDC‐E2. As expected, 90 of 90 previously known AMA‐positive patients remained positive with this assay but, interestingly, 20% of the rigorously defined AMA‐negative patient group had antibodies to one or more of the mitochondrial autoantigens. Furthermore, 100% of these newly detected AMA‐positive patients were anti‐nuclear antibody (ANA) positive. Conclusion: The development of this assay reflects the potential for automated detection with rapid and reliable assaying and further highlights the diminished number of truly AMA‐negative PBC patients. (HEPATOLOGY 2007;45:659–665.)
Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the ...immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.
Homo‐ and heteroleptic bismuth thiolato complexes have been synthesised and characterised from biologically relevant tetrazole‐, imidazole‐, thiadiazole‐ and thiazole‐based heterocyclic thiones ...(thiols): 1‐methyl‐1H‐tetrazole‐5‐thiol (1‐MMTZ(H)); 4‐methyl‐4H‐1,2,4‐triazole‐3‐thiol (4‐MTT(H)); 1‐methyl‐1H‐imidazole‐2‐thiol (2‐MMI(H)); 5‐methyl‐1,3,4‐thiadiazole‐2‐thiol (5‐MMTD(H)); 1,3,4‐thiadiazole‐2‐dithiol (2,5‐DMTD(H)2); and 4‐(4‐bromophenyl)thiazole‐2‐thiol (4‐BrMTD(H)). Reaction of BiPh3 with 1‐MMTZ(H) produced the rare BiV thiolato complex BiPh(1‐MMTZ)4, which undergoes reduction in DMSO to give BiPh(1‐MMTZ)2{(1‐MMTZ(H)}2. Reactions with PhBiCl2 or BiPh3 generally produced monophenylbismuth thiolates, BiPh(SR)2. The crystal structures of BiPh(1‐MMTZ)2{1‐MMTZ(H)}2, BiPh(5‐MMTD)2, BiPh{2,5‐DMTD(H)}2(Me2CO) and Bi(4‐BrMTD)3 were obtained. Evaluation of the bactericidal properties against M. smegmatis, S. aureus, MRSA, VRE, E. faecalis and E. coli showed complexes containing the anionic ligands 1‐ MMTZ, 4‐MTT and 4‐BrMTD to be most effective. The dithiolato dithione complexes BiPh(4‐MTT)2{4‐MTT(H)}2 and BiPh(1‐MMTZ)2{1‐MMTZ(H)}2 were most effective against all the bacteria: MICs 0.34 μM for BiPh(4‐MTT)2{4‐MTT(H)}2 against VRE, and 1.33 μM for BiPh(1‐MMTZ)2{1‐MMTZ(H)}2 against M. smegmatis and S. aureus. Tris‐thiolato BiIII complexes were least effective overall. All complexes showed little or no toxicity towards mammalian COS‐7 cells at 20 μg mL−1.
Bismuth takes on bacteria: Thirteen new homo‐ and heteroleptic bismuth thiolato complexes; BiPh(SR)2, BiPh(SR)2(HSR)2 and Bi(SR)3, derived from a series of biologically relevant heterocyclic thiols (thiones), demonstrate excellent antibacterial activities (see figure).
The high mortality of Plasmodium falciparum malaria is the result of a parasite ligand, PfEMP1 (P. falciparum) erythrocyte membrane protein 1), on the surface of infected red blood cells (IRBCs), ...which adheres to the vascular endothelium and causes the sequestration of IRBCs in the microvasculature. PfEMP1 transport to the IRBC surface involves Maurer's clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurer's cleft protein, SBP1 (skeleton-binding protein 1), prevented IRBC adhesion because of the loss of PfEMP1 expression on the IRBC surface. PfEMP1 was still present in Maurer's clefts, and the transport and localization of several other Maurer's cleft proteins were unchanged. Maurer's clefts were altered in appearance and were no longer found as close to the periphery of the IRBC. Complementation of mutant parasites with sbp1 led to the reappearance of PfEMP1 on the IRBC surface and the restoration of adhesion. Our results demonstrate that SBP1 is essential for the translocation of PfEMP1 onto the surface of IRBCs and is likely to play a pivotal role in the pathogenesis of P. falciparum malaria.
Five mixed thiolatobismuth(III) complexes BiPh(5‐MMTD)2{4‐MMT(H)} (1), Bi(1‐MMTZ)2{(PYM)(PYM(H))2} (2), Bi(MBT)2(5‐MMTD) (3), Bi(4‐BrMTD)3{2‐MMI(H)} (4) and Bi(1‐MMTZ)2{1‐MMTZ(H)}(2‐MMI){2‐MMI(H)2} ...(5) were synthesised from imidazole‐, thiazole‐, thiadiazole‐, triazole‐, tetrazole‐ and pyrimidine‐based heterocyclic thiones. Four of these complexes 1–4 were synthesized from BiPh3, while complex 5 was obtained from Bi4‐(MeO)Ph3. Complexes 1–5 were structurally characterised by XRD. Evaluation of the antibacterial properties against Mycobacterium smegmatis, Staphylococcus aureus, Methicillin‐resistant S. aureus (MRSA), Vancomycin‐resistant Enterococcus (VRE), Enterococcus faecalis and Escherichia coli showed that mixed thiolato complexes containing the anionic thiazole‐based ligands MBT and 4‐BrMTD are most effective. The mixed thiolato complexes Bi(MBT)2(5‐MMTD) (3) having thiazole‐ and thiadiazole‐ and Bi(4‐BrMBT)3{2‐MMI(H)} (4) containing thiazole‐ and imidazole‐based ligands proved to be more efficient, with low minimum inhibitory concentrations of 1.73 and 3.45 µm for 3 against VRE and E. faecalis, respectively, and 2.20 µm for 4 against M. smegmatis and E. faecalis. All complexes showed little or no toxicity towards mammalian COS‐7 cell lines at 20 µg mL–1.
Bismuth(III) mixed thiolato complexes BiPh(5‐MMTD)2{4‐MMT(H)} (1), Bi(1‐MMTZ)2{(PYM)(PYM(H))2} (2), Bi(MBT)2(5‐MMTD) (3), Bi(4‐BrMTD)3{2‐MMI(H)} (4) and Bi(1‐MMTZ)2{1‐MMTZ(H)}(2‐MMI){2‐MMI(H)2} (5) were synthesised, and their antibacterial activity was studied. Heteroleptic complexes seem to have improved activity.
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