Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry.

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Nociceptive Sensory Neurons Mediate Inflammation Induced by Bacillus Anthracis Edema Toxin.

Bacterial products are able to act on nociceptive neurons during pathogenic infection. Neurogenic inflammation is an active part of pain signaling and has recently been shown to impact host-pathogen defense. Bacillus anthracis Edema Toxin (ET) produces striking edema in peripheral tissues, but the cellular mechanisms involved in tissue swelling are not completely understood. Here, we find that nociceptive neurons play a role in ET-induced edema and inflammation in mice. Subcutaneous footpad infection of B. anthracis Sterne caused ET-dependent local mechanical allodynia, paw swelling and body weight gain. Subcutaneous administration of ET induced paw swelling and vascular leakage, the early phases of which were attenuated in the absence of Trpv1+ or Nav1.8+ nociceptive neurons. Nociceptive neurons express the anthrax toxin receptor ANTXR2, but this did not mediate ET-induced edema. ET induced local cytokine expression and neutrophil recruitment, which were dependent in part on Trpv1+ nociceptive neurons. Ablation of Trpv1+ or Nav1.8+ nociceptive neurons also attenuated early increases in paw swelling and body weight gain during live B. anthracis infection. Our findings indicate that nociceptive neurons play an active role in inflammation caused by B. anthracis and Edema Toxin to potentially influence bacterial pathogenesis.

HDAC8 Activates AKT through Upregulating PLCB1 and Suppressing DESC1 Expression in MEK1/2 Inhibition-Resistant Cells.

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-ß1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.

Bacillus anthracis edema toxin inhibits hypoxic pulmonary vasoconstriction via edema factor and cAMP-mediated mechanisms in isolated perfused rat lungs.

Bacillus anthracis edema toxin (ET) inhibited lethal toxin-stimulated pulmonary artery pressure (Ppa) and increased lung cAMP levels in our previous study. We therefore examined whether ET inhibits hypoxic pulmonary vasoconstriction (HPV). Following baseline hypoxic measures in isolated perfused lungs from healthy rats, compared with diluent, ET perfusion reduced maximal Ppa increases (mean ± SE percentage of maximal Ppa increase with baseline hypoxia) during 6-min hypoxic periods (FIO2 = 0%) at 120 min (16 ± 6% vs. 51 ± 6%, P = 0.004) and 180 min (11.4% vs. 55 ± 6%, P = 0.01). Protective antigen-mAb (PA-mAb) and adefovir inhibit host cell edema factor uptake and cAMP production, respectively. In lungs perfused with ET following baseline measures, compared with placebo, PA-mAb treatment increased Ppa during hypoxia at 120 and 180 min (56 ± 6% vs. 10 ± 4% and 72 ± 12% vs. 12 ± 3%, respectively, P ≤ 0.01) as did adefovir (84 ± 10% vs. 16.8% and 123 ± 21% vs. 26 ± 11%, respectively, P ≤ 0.01). Compared with diluent, lung perfusion with ET for 180 min reduced the slope of the relationships between Ppa and increasing concentrations of endothelin-1 (ET-1) (21.12 ± 2.96 vs. 3.00 ± 0.76 × 108 cmH2O/M, P < 0.0001) and U46619, a thromboxane A2 analogue (7.15 ± 1.01 vs. 3.74 ± 0.31 × 107 cmH2O/M, P = 0.05) added to perfusate. In lungs isolated from rats after 15 h of in vivo infusions with either diluent, ET alone, or ET with PA-mAb, compared with diluent, the maximal Ppa during hypoxia and the slope of the relationship between change in Ppa and ET-1 concentration added to the perfusate were reduced in lungs from animals challenged with ET alone (P ≤ 0.004) but not with ET and PA-mAb together (P ≥ 0.73). Inhibition of HPV by ET could aggravate hypoxia during anthrax pulmonary infection.NEW & NOTEWORTHY The most important findings here are edema toxin's potent adenyl cyclase activity can interfere with hypoxic pulmonary vasoconstriction, an action that could worsen hypoxemia during invasive anthrax infection with lung involvement. These findings, coupled with other studies showing that lethal toxin can disrupt pulmonary vascular integrity, indicate that both toxins can contribute to pulmonary pathophysiology during infection. In combination, these investigations provide a further basis for the use of antitoxin therapies in patients with worsening invasive anthrax disease.

Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality.

Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85ß (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.

Anthrax Edema and Lethal Toxins Differentially Target Human Lung and Blood Phagocytes.

Bacillus anthracis, the causative agent of inhalation anthrax, is a serious concern as a bioterrorism weapon. The vegetative form produces two exotoxins: Lethal toxin (LT) and edema toxin (ET). We recently characterized and compared six human airway and alveolar-resident phagocyte (AARP) subsets at the transcriptional and functional levels. In this study, we examined the effects of LT and ET on these subsets and human leukocytes. AARPs and leukocytes do not express high levels of the toxin receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2). Less than 20% expressed surface TEM8, while less than 15% expressed CMG2. All cell types bound or internalized protective antigen, the common component of the two toxins, in a dose-dependent manner. Most protective antigen was likely internalized via macropinocytosis. Cells were not sensitive to LT-induced apoptosis or necrosis at concentrations up to 1000 ng/mL. However, toxin exposure inhibited B. anthracis spore internalization. This inhibition was driven primarily by ET in AARPs and LT in leukocytes. These results support a model of inhalation anthrax in which spores germinate and produce toxins. ET inhibits pathogen phagocytosis by AARPs, allowing alveolar escape. In late-stage disease, LT inhibits phagocytosis by leukocytes, allowing bacterial replication in the bloodstream.

BPTES inhibits anthrax lethal toxin-induced inflammatory response.

Bacillus anthracis is a lethal agent of anthrax disease and the toxins are required in anthrax pathogenesis. The anthrax lethal toxin can trigger NLRP1b inflammasome activation and pyroptosis. Although the underlying mechanism is well understood, the medications targeting the NLRP1b inflammasome are not available in the clinic. Herein, we describe that BPTES, a known Glutaminase (GLS) inhibitor, is an effective NLRP1b inflammasome inhibitor. BPTES could effectively and specifically suppress NLRP1b inflammasome activation in macrophages but have no effects on NLRP3, NLRC4 and AIM2 inflammasome activation. Mechanistically, BPTES alleviated the UBR2 mediated proteasomal degradation pathway of the NLRP1b N terminus, thus blocking the release of the CARD domain for subsequent caspase-1 processing. Furthermore, BPTES could prevent disease progression in mice challenged with the anthrax lethal toxin. Taken together, our studies indicate that BPTES can be a promising pharmacological inhibitor to treat anthrax lethal toxin-related inflammatory diseases.

Potential Therapeutic Targeting of Coronavirus Spike Glycoprotein Priming.

Processing of certain viral proteins and bacterial toxins by host serine proteases is a frequent and critical step in virulence. The coronavirus spike glycoprotein contains three (S1, S2, and S2') cleavage sites that are processed by human host proteases. The exact nature of these cleavage sites, and their respective processing proteases, can determine whether the virus can cross species and the level of pathogenicity. Recent comparisons of the genomes of the highly pathogenic SARS-CoV2 and MERS-CoV, with less pathogenic strains (e.g., Bat-RaTG13, the bat homologue of SARS-CoV2) identified possible mutations in the receptor binding domain and in the S1 and S2' cleavage sites of their spike glycoprotein. However, there remains some confusion on the relative roles of the possible serine proteases involved for priming. Using anthrax toxin as a model system, we show that in vivo inhibition of priming by pan-active serine protease inhibitors can be effective at suppressing toxicity. Hence, our studies should encourage further efforts in developing either pan-serine protease inhibitors or inhibitor cocktails to target SARS-CoV2 and potentially ward off future pandemics that could develop because of additional mutations in the S-protein priming sequence in coronaviruses.

Frontline Science: Anthrax lethal toxin-induced, NLRP1-mediated IL-1ß release is a neutrophil and PAD4-dependent event.

Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better-studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming-independent LT-induced release of IL-1ß in mice within hours of toxin challenge. We find IL-1ß release to be a NLRP1b- and caspase-1-dependent, NLRP3 and caspase-11-independent event that requires both neutrophils and peptidyl arginine deiminiase-4 (PAD4) activity. The simultaneous LT-induced IL-18 response is neutrophil-independent. Bone marrow reconstitution experiments in mice show toxin-induced IL-1ß originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1ß, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1ß, they were unable to bind or endocytose LT and did not secrete IL-1ß in response to the toxin. LT-treated mice had higher levels of cell-free DNA and HMGB1 in circulation than PBS-treated controls, and treatment of mice with recombinant DNase reduced the neutrophil- and NLRP1-dependent IL-1ß release. DNA sensor AIM2 deficiency, however, did not impact IL-1ß release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell-free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT-treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin.

Engineering of an Iranian Bordetella pertussis strain producing inactive pertussis toxin.

Introduction. Differences between the genomic and virulence profile of Bordetella pertussis circulating strains and vaccine strains are considered as one of the important reasons for the resurgence of whooping cough (pertussis) in the world. Genetically inactivated B. pertussis is one of the new strategies to generate live-attenuated vaccines against whooping cough.Aim. The aim of this study was to construct a B. pertussis strain based on a predominant profile of circulating Iranian isolates that produces inactivated pertussis toxin (PTX).Methodology. The B. pertussis strain BPIP91 with predominant genomic and virulence pattern was selected from the biobank of the Pasteur Institute of Iran. A BPIP91 derivative with R9K and E129G alterations in the S1 subunit of PTX (S1mBPIP91) was constructed by the site-directed mutagenesis and homologous recombination. Genetic stability and antigen expression of S1mBPIP91 were tested by serially in vitro passages and immunoblot analyses, respectively. The reduction in toxicity of S1mBPIP91 was determined by Chinese hamster ovary (CHO) cell clustering.Results. All constructs and S1mBPIP91 were confirmed via restriction enzyme analysis and DNA sequencing. The engineered mutations in S1mBPIP91 were stable after 20 serial in vitro passages. The production of virulence factors was also confirmed in S1mBPIP91. The CHO cell-clustering test demonstrated the reduction in PTX toxicity in S1mBPIP91.Conclusion. A B. pertussis of the predominant genomic and virulence lineage in Iran was successfully engineered to produce inactive PTX. This attenuated strain will be useful to further studies to develop both whole cell and acellular pertussis vaccines.

Different mechanisms of two anti-anthrax protective antigen antibodies and function comparison between them.

BACKGROUND: Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. METHODS: The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. RESULTS: In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. CONCLUSIONS: Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.

Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling.

Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.

Repeat-Dose Toxicity Study of a Lyophilized Recombinant Protective Antigen-Based Anthrax Vaccine Adjuvanted With CpG 7909.

A recombinant protective antigen (rPA) anthrax vaccine candidate (rPA7909) was developed as a next-generation vaccine indicated for postexposure prophylaxis of disease resulting from suspected or confirmed Bacillus anthracis exposure. The lyophilized form of rPA7909-vaccinated candidate contains 75 µg purified rPA, 750 µg aluminum (as Alhydrogel adjuvant), and 250 µg of an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide CpG 7909 in a 0.5 mL phosphate-buffered suspension. General toxicity and local reactogenicity were evaluated in Sprague Dawley rats vaccinated with the full human dose of rPA7909 by intramuscular injection. Animals were immunized on study days 1, 15, and 29. Control groups were administered diluent only or adjuvant control (excipients, CpG 7909, and Alhydrogel adjuvant in diluent) intramuscularly at the same dose volume and according to the same schedule used for rPA7909. Toxicity was assessed based on the results of clinical observations, physical examinations, body weights, injection site reactogenicity, ophthalmology, clinical pathology (hematology, coagulation, and serum chemistry), organ weights, and macroscopic and microscopic pathology evaluation. The immune response to rPA7909 vaccination was confirmed by measuring serum anti-PA immunoglobulin G levels. The rPA7909 vaccine produced no apparent systemic toxicity and only transient reactogenicity at the injection site. The injection site reaction from animals receiving the adjuvant control was very similar to those receiving rPA7909 with respect to the inflammation. The inflammatory response observed in the injection site and the draining lymph nodes was consistent with expected immune stimulation. The overall results indicated a favorable safety profile for rPA7909.

Classification of Helicobacter pylori Virulence Factors: Is CagA a Toxin or Not?

Since its discovery, Helicobacter pylori has been identified as the causative agent of various gastric diseases. H. pylori produces myriads of disease-associated virulence factors. These bacterial determinants can be distinguished as cell-binding factors, immunoregulatory components, survival factors, toxins, and effector proteins. For most of these factors there is consensus about their classification. However, there is a strong dispute in the literature as to whether one of the best-studied factors, CagA, represents a toxin or not. CagA displays unique functions that are clearly different from conventional toxins, and CagA counteracts the activities of an established H. pylori toxin, VacA. Canonical toxins commonly have specific (and narrow) targets, can act even in the absence of the bacterial cell, and elicit acute damage to host cells. However, there is still no agreement on the classification of CagA. Here we discuss whether CagA acts as a toxin, and propose a classification consensus for CagA.

Group A streptococcal M-protein specific antibodies and T-cells drive the pathology observed in the rat autoimmune valvulitis model.

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune mediated diseases triggered by group A streptococcal (GAS) infections. Molecular mimicry between GAS M-proteins and host tissue proteins has been proposed as the mechanism that initiates autoreactive immune responses in ARF/RHD. However, the individual role of antibodies and T-cells specific for GAS M-proteins in the pathogenesis of autoimmune carditis remains under-explored. The current study investigated the role of antibodies and T-cells in the development of carditis in the Lewis rat autoimmune valvultis (RAV) model by transferring serum and/or splenic T-cells from rats previously injected with GAS recombinant M5 protein. Here we report that serum antibodies alone and serum plus in vitro expanded rM5-specific T-cells from hyperimmune rats were capable of transferring carditis to naïve syngeneic animals. Moreover, the rats that received combined serum and T-cells developed more severe carditis. Recipient rats developed mitral valvulitis and myocarditis and showed prolongation of P-R intervals in electrocardiography. GAS M5 protein-specific IgG reactivity and T-cell recall response were also demonstrated in recipient rats indicating long-term persistence of antibodies and T-cells following transfer. The results suggest that both anti-GAS M5 antibodies and T-cells have differential propensity to induce autoimmune mediated carditis in syngeneic rats following transfer. The results highlight that antibodies and effector T-cells generated by GAS M protein injection can also independently home into cardiac tissue to cross-react with tissue proteins causing autoimmune mediated immunopathology.

Diverse small molecules prevent macrophage lysis during pyroptosis.

Pyroptosis is a programmed process of proinflammatory cell death mediated by caspase-1-related proteases that cleave the pore-forming protein, gasdermin D, causing cell lysis and release of inflammatory intracellular contents. The amino acid glycine prevents pyroptotic lysis via unknown mechanisms, without affecting caspase-1 activation or pore formation. Pyroptosis plays a critical role in diverse inflammatory diseases, including sepsis. Septic lethality is prevented by glycine treatment, suggesting that glycine-mediated cytoprotection may provide therapeutic benefit. In this study, we systematically examined a panel of small molecules, structurally related to glycine, for their ability to prevent pyroptotic lysis. We found a requirement for the carboxyl group, and limited tolerance for larger amino groups and substitution of the hydrogen R group. Glycine is an agonist for the neuronal glycine receptor, which acts as a ligand-gated chloride channel. The array of cytoprotective small molecules we identified resembles that of known glycine receptor modulators. However, using genetically deficient Glrb mutant macrophages, we found that the glycine receptor is not required for pyroptotic cytoprotection. Furthermore, protection against pyroptotic lysis is independent of extracellular chloride conductance, arguing against an effect mediated by ligand-gated chloride channels. Finally, we conducted a small-scale, hypothesis-driven small-molecule screen and identified unexpected ion channel modulators that prevent pyroptotic lysis with increased potency compared to glycine. Together, these findings demonstrate that pyroptotic lysis can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.

The involvement of NF-κB/P38 pathways in Scutellaria baicalensis extracts attenuating of Escherichia coli K88-induced acute intestinal injury in weaned piglets.

The present study was carried out to evaluate the effect of dietary supplementation of Scutellaria baicalensis extracts (SBE) on intestinal health in terms of morphology, barrier integrity and immune responses in weaned piglets challenged with Escherichia coli K88. A total of seventy-two weaned piglets were assigned into two groups to receive a basal diet without including antibiotic additives or the basal diet supplemented 1000 mg SBE/kg diet for 14 d. On day 15, twelve healthy piglets from each group were selected to expose to oral administration of either 10 ml 1 × 109 colony-forming units of E. coli K88 or the vehicle control. After 48 h of E.coli K88 challenge, blood was sampled, and then all piglets were killed humanely for harvesting jejunal and ileal samples. Dietary supplementation of SBE significantly decreased diarrhoea frequency and improved feed conversion ratio (P < 0·05). SBE supplementation to E.coli K88-challenged piglets improved villous height and villous height/crypt depth (P < 0·05), recovered the protein expression of occludin and zonula occludens-2 in both the jejunum and ileum (P < 0·05), and mitigated the increases in plasma IL-1ß, TNF-α, IL-6, IgA and IgG (P < 0·05). Meanwhile, dietary SBE effectively inhibited the stimulation of NF-κB, P38 and TNF-α as well as IL-1ß in the small intestine of piglets challenged by E. coli K88 and prevented the activation of NF-κB/P38 signalling pathways (P < 0·05). Collectively, SBE supplementation can potently attenuate diarrhoea in weaning piglets and decrease inflammatory cytokine expressions through inhibiting the NF-κB and P38 signalling pathways.

Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death and reduced immunoglobulin release.

Streptococcal pyrogenic exotoxin (Spe) A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage to Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leucocyte profile of tonsil is unique, the impact of SpeA production on human tonsil cell function was investigated. Human tonsil cells from routine tonsillectomy were co-incubated with purified streptococcal superantigens or culture supernatants from isogenic streptococcal isolates, differing only in superantigen production. Tonsil cell proliferation was quantified by tritiated thymidine incorporation, and cell surface characteristics assessed by flow cytometry. Soluble mediators including immunoglobulin were measured using enzyme-linked immunosorbent assay. Tonsil T cells proliferated in response to SpeA and demonstrated typical release of proinflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7 days. In contrast, marked B cell apoptosis and abrogation of total immunoglobulin (Ig)A, IgM, and IgG production occurred in the presence of SpeA and other superantigens. In SpeA-stimulated cultures, T follicular helper (Tfh) cells showed a reduction in C-X-C chemokine receptor (CXCR)5 (CD185) expression, but up-regulation of OX40 (CD134) and inducible T cell co-stimulator (ICOS) (CD278) expression. The phenotypical change in the Tfh population was associated with impaired chemotactic response to CXCL13. SpeA and other superantigens cause dysregulated tonsil immune function, driving T cells from Tfh to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a probable survival advantage.

Bacillus anthracis lethal toxin, but not edema toxin, increases pulmonary artery pressure and permeability in isolated perfused rat lungs.

Although lethal toxin (LT) and edema toxin (ET) contribute to lethality during Bacillus anthracis infection, whether they increase vascular permeability and the extravascular fluid accumulation characterizing this infection is unclear. We employed an isolated perfused Sprague-Dawley rat lung model to investigate LT and ET effects on pulmonary vascular permeability. Lungs (n ≥ 6 per experimental group) were isolated, ventilated, suspended from a force transducer, and perfused. Lung weight and pulmonary artery (Ppa) and left atrial pressures were measured over 4 h, after which pulmonary capillary filtration coefficients (Kf.c) and lung wet-to-dry weight ratios (W/D) were determined. When compared with controls, LT increased Ppa over 4 h and Kf.c and W/D at 4 h (P < 0.0001). ET decreased Ppa in a significant trend (P = 0.09) but did not significantly alter Kf.c or W/D (P ≥ 0.29). Edema toxin actually blocked LT increases in Ppa but not LT increases in Kf.c and W/D. When Ppa was maintained at control levels, LT still increased Kf.c and W/D (P ≤ 0.004). Increasing the dose of each toxin five times significantly increased and a toxin-directed monoclonal antibody decreased the effects of each toxin (P ≤ 0.05). Two rho-kinase inhibitors (GSK269962 and Y27632) decreased LT increases in Ppa (P ≤ 0.02) but actually increased Kf.c and W/D in LT and control lungs (P ≤ 0.05). A vascular endothelial growth factor receptor inhibitor (ZM323881) had no significant effect (P ≥ 0.63) with LT. Thus, LT but not ET can increase pulmonary vascular permeability independent of increased Ppa and could contribute to pulmonary fluid accumulation during anthrax infection. However, pulmonary vascular dilation with ET could disrupt protective hypoxic vasoconstriction. NEW & NOTEWORTHY The most important findings from the present study are that Bacillus anthracis lethal toxin increases pulmonary artery pressure and pulmonary permeability independently in the isolated rat lung, whereas edema toxin decreases the former and does not increase permeability. Each effect could be a basis for organ dysfunction in patients with this lethal infection. These findings further support the need for adjunctive therapies that limit the effects of both toxins during infection.

A new triple chimeric protein as a high immunogenic antigen against anthrax toxins: theoretical and experimental analyses.

Background: Anthrax is a zoonotic disease caused by Bacillus anthracis and it can be deadly in 6 days. Considerable efforts have been conducted toward developing more effective veterinary and human anthrax vaccines because these common vaccines have several limitations. B. anthracis secretes a tripartite toxin, comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). Several studies have shown important role of PA in protection of anthrax. LF and EF induce production of toxin neutralizing antibodies too. PA in fusion form with LF/EF has synergistic effects as a potential subunit vaccine. Methods: In this study, for the first time, a triple chimeric protein called ELP was modeled by fusing three different domains of anthrax toxic antigens, the N-terminal domains of EF and LF, and the C-terminal domain of PA as a high immunogenic antigen using Modeller 9.19 software. Immunogenicity of the ELP was assessed in guinea pigs using enzyme-linked immunosorbent assay (ELISA) test and MTT assay. Results: Theoretical studies and molecular dynamics (MD) simulation results suggest that the ELP model had acceptable quality and stability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified ELP, its domains, and PA were matched with their molecular size and confirmed by western blotting analysis. In the immune guinea pigs, antibody was produced against all of the ELP domains. It was observed that ELP induced strong humoral response and could protect murine macrophage cell line (RAW 264.7 cells) against anthrax lethal toxin (LeTx). Conclusions: ELP chimeric antigen could be considered as a high immunogenic antigen.