The mucosal immune system serves as the first line of defense against Bordetella pertussis. Intranasal vaccination, due to its potential to induce systemic and mucosal immune responses, appears to prevent the initial adherence and colonization of the bacteria at the first point of contact. In the present study, two B. pertussis antigens, pertussis Toxoid (PTd) and Filamentous hemagglutinin (FHA), which play a very significant role in virulence and protection against pertussis, were encapsulate into N-trimethyl chitosan (TMC) nanoparticulate systems. After preparation of TMC nanoparticles (NPs), the NPs were characterized and their ability to induce efficient immune responses against B. pertussis was studied in a mouse model. Our findings showed that PTd + FHA-loaded TMC NPs have strong ability to induce IL-4, IL-17, IFN-γ, IgG, and IgA in the mouse model. Results from this study suggest that nasal administration of the PTd + FHA-loaded TMC NPs induced not only a systemic immune response but also a local mucosal response, which may improve the efficacy of pertussis prevention through respiratory tract transmission.
The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis
0.05% Trypsin-EDTA (1x) Life Technologies Cat#25300-054 MyHCa614-629 GenScript Cat#639666-1 Protease, Type XIV: Bacterial, From Streptomyces griseus Sigma-Aldrich Cat#P5147-5G Deoxyribonuclease I Worthington Cat#LS002139 Collagenase II Worthington Cat#LS004177 Pertussis Toxin from Bordetella pertussis List Biological Laboratories, Inc. Cat#180, #181 Mycobacterium Tuberculosis Des. H37 Ra BD 231141 L-Glutamine Corning Inc. Cat#25-005-CI
- refractometer at 37°C with a flow rate of 41 μL/min. Full-length CtxA1, full-length PtxS1 (List Biologicals, Campbell, CA), or peptide (Peptide 2.0, Chantilly, VA) was amide-coupled well plates were incubated with 1 μg of Ptx (List Biologicals) in serum-free Ham's F-12
Cholera toxin (Ctx) is an AB-type protein toxin that acts as an ADP-ribosyltransferase to disrupt intracellular signaling in the target cell. It moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. The catalytic CtxA1 subunit then dissociates from the rest of the toxin, unfolds, and activates the ER-associated degradation system for export to the cytosol. Translocation occurs through an unusual ratchet mechanism in which the cytosolic chaperone Hsp90 couples CtxA1 refolding with CtxA1 extraction from the ER. Here, we report that Hsp90 recognizes two peptide sequences from CtxA1: an N-terminal RPPDEI sequence (residues 11-16) and an LDIAPA sequence in the C-terminal region (residues 153-158) of the 192 amino acid protein. Peptides containing either sequence effectively blocked Hsp90 binding to full-length CtxA1. Both sequences were necessary for the ER-to-cytosol export of CtxA1. Mutagenesis studies further demonstrated that the RPP residues in the RPPDEI motif are required for CtxA1 translocation to the cytosol. The LDIAPA sequence is unique to CtxA1, but we identified an RPPDEI-like motif at the N- or C-termini of the A chains from four other ER-translocating toxins that act as ADP-ribosyltransferases: pertussis toxin, Escherichia coli heat-labile toxin, Pseudomonas aeruginosa exotoxin A, and Salmonella enterica serovar Typhimurium ADP-ribosylating toxin. Hsp90 plays a functional role in the intoxication process for most, if not all, of these toxins. Our work has established a defined RPPDEI binding motif for Hsp90 that is required for the ER-to-cytosol export of CtxA1 and possibly other toxin A chains as well. This article is protected by copyright. All rights reserved.
Glutaminase 1 Inhibition Reduces Glycolysis and Ameliorates Lupus-like Disease in MRL/lpr Mice and Experimental Autoimmune Encephalomyelitis
- tuberculosis extract (H37Ra; Difco), distributed between the two hind flanks. On days 0 and 2, 150 ng/mouse pertussis toxin (List Biological Laboratories) was given by intraperitoneal injection. Mice were monitored and weighted daily until day 28. Following clinical
Glutaminase 1 (Gls1) is the first enzyme in glutaminolysis. The selective Gls1 inhibitor Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) suppresses Th17 development and ameliorates experimental autoimmune encephalomyelitis (EAE). However, whether inhibition of glutaminolysis is beneficial for the treatment of systemic lupus erythematosus (SLE) and the involved mechanisms are still unknown. MRL/lpr mice were treated by BPTES or vehicle control and disease activity was examined. Then naïve CD4+ T cells from patients with SLE were cultured under Th17 conditions with BPTES or the vehicle. Furthermore, using newly generated Gls1 conditional knockout mice in IL-17 producing cells, in vitro Th17 differentiation were examined and EAE was induced in these mice. Glutaminolysis and glycolysis were measured by extracellular flux analyzer. The expression of hypoxia-inducible factor 1α (HIF1α) was also examined by Western blotting. Treatment of MRL/lpr mice with BPTES improved autoimmune pathology in a Th17-dependent fashion. T cells from patients with SLE treated with BPTES display decreased Th17 differentiation (P < 0.05). Using the conditional knockout mice we demonstrate that both the in vitro Th17 differentiation (P < 0.05) and the development of EAE depend on Gls1. Gls1 inhibition reduced glycolysis and the expression of HIF1α protein which induces glycolysis. We have demonstrated that inhibition of glutaminolysis represents a potential new treatment strategy for patients with SLE and Th17-related autoimmune diseases. Mechanistically we have shown that inhibition of glutaminolysis affects the glycolysis pathway by reducing Hif1α protein in Th17 cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Gut microbiota depletion from early adolescence alters adult immunological and neurobehavioral responses in a mouse model of multiple sclerosis
; - The emulsion was injected subcutaneously in both hind flanks of each animal. The mice were intraperitoneally treated with 300 ng of pertussis toxin from bordetella pertussis (dissolved in 100 μl PBS; List Biological Lab, USA) at the time of immunization and again 48