We describe the molecular mode of action and pharmacodynamics of a new molecular entity (NME) that induces the NLRP3 inflammasome-mediated innate immune response. This innate response reduces the pathogen load in an experimentally induced methicillin-resistant Staphylococcos aureus infection, enhances survival in an experimentally induced Gram-negative bacteremia, and overrides the escape mechanism of an obligate intracellular pathogen, viz. Chlamydia pneumoniae. Furthermore, the NME is more effective than standard-of-care antibiotic therapy in a clinically established multifactorial bacterial infection. Analysis of transcriptional regulation of inflammasome signaling genes and innate/adaptive immune genes revealed consistent and significant host changes responsible for the improved outcomes in these infections. These studies pave the way for the development of first-in-class drugs that enhance inflammasome-mediated pathogen clearance and identify the NLRP3 inflammasome as a drug target to address the global problem of emerging new infectious diseases and the reemergence of old diseases in an antibiotic-resistant form.
Differentiation between species of similar biological structure is of critical importance in biosensing applications. Here, we report specific detection of Bacillus anthracis (BA) spores from that of close relatives, such as B. thuringiensis (BT), B. cereus (BC), and B. subtilis (BS) by varying the flow speed of the sampling liquid over the surface of a piezoelectric microcantilever sensor (PEMS). Spore binding to the anti-BA spore IgG coated PEMS surface is determined by monitoring the resonance frequency change in the sensor's impedance vs. frequency spectrum. Flow increases the resonance frequency shift at lower flow rates until the impingement force from the flow overcomes the binding strength of the antigen and decreases the resonance frequency shift at higher flow rates. We showed that the change from increasing to decreasing resonance frequency shift occurred at a lower fluid flow speed for BT, BC, and BS spores than for BA spores. This trend reduces the cross reactivity ratio of BC, BS, and BT to the anti-BA spore IgG immobilized PEMS from around 0.4 at low flow velocities to less than 0.05 at 3.8 mm s −1 . This cross reactivity ratio of 0.05 was essentially negligible considering the experimental uncertainty. The use of the same flow that is used for detection to further distinguish the specific binding (BA to anti-BA spore antibody) from nonspecific binding (BT, BC, and BS to anti-BA spore antibody) is unique and has great potential in the detection of general biological species.
Experiments were undertaken to isolate a component of the serum of goat (Capra hircus) that is effective at mediating an innate immune response. This report describes the isolation and structure elucidation of 1-(N-acetyl-ALYDKGYTSKEQKDCVGI)-2-arachidonoyl-3-stearoyl glyceride (1) and its immunomodulatory activity. A dose-response relationship for inflammatory cytokine and chemokine production and release from human fibroblasts incubated with nanomolar concentrations of 1 was shown. Moreover, the membrane transport role of the diacylglycerol moiety in 1 is demonstrated with nanomolar quantities of the transfected N-acetyl peptide moiety of 1 also inducing inflammatory cytokine and chemokine production and release. The apparent EC99 for 1 was 3 ng/mL (1 nM). The likely biological role for naturally occurring 1 as a damage-associated molecular pattern is postulated.
An array of three identical piezoelectric microcantilever sensors ͑PEMSs͒ consisting of a lead zirconate titanate layer bonded to a glass layer was fabricated and examined for simultaneous, in situ, real-time, all-electrical detection of Bacillus anthracis ͑BA͒ spores in an aqueous suspension using the first longitudinal extension mode of resonance. With anti-BA antibody immobilized on the sensor surfaces all three PEMS exhibited identical BA detection resonance frequency shifts at all tested concentrations, 10-10 7 spores/ ml with a standard deviation of less than 10%. The detection concentration limit of 10 spores/ml was about two orders of magnitude lower than would be permitted by flexural peaks. In blinded-sample testing, the array PEMS detected BA in three samples containing BA: ͑1͒ 3.3ϫ 10 3 spores/ ml, ͑2͒ a mixture of 3.3ϫ 10 3 spores/ ml and 3.3 ϫ 10 5 S. aureus ͑SA͒ and P. aeruginosa ͑PA͒ per ml, and ͑3͒ a mixture of 3.3ϫ 10 3 spores/ ml with 3.3ϫ 10 6 SA+ PA/ ml. There was no response to a sample containing only 3.3ϫ 10 6 SA+ PA/ ml. These results illustrate the sensitivity, specificity, reusability, and reliability of array PEMS for in situ, real-time detection of BA spores.
The present paper reports a bio-computational study carried out with the aim of understanding the binding mode of anti-TB herbal ligands onto the homology modeled structure of fatty acid synthase of Mycobacterium tuberculosis (M.tb) H37Rv. Sequence alignment of beta-ketoacyl ACP synthase (KAS) domain of the protein with other related KAS sequences of PDB database revealed high degree of sequence variation. However, the catalytic triad comprising of CHH (cys150-his279-his320) was found to be conserved in the KAS sequence of M.tb H37Rv. The tertiary structure of this protein predicted using genetic algorithm operator in the MODELLER package appeared to give a satisfactory structure for the purpose of studying ligand and substrate binding pockets on the protein. PDB templates complexed with ligands (citric acid and lauric acid) were used for model building. Docking studies carried out with different herbal ligands suggest that, aloe-emodin and nimbin are the best herbal candidates to replace the synthetic drugs 'thiolactomycin/cerulenin'.
We reported an endogenously derived inflammasome activating lipopeptide (1-peptidyl-2-arachidonoyl-3-stearoyl glyceride) with the amino acid sequence for the peptide being acALYDKGYTSKEQKDCVGI (acALY18) and determined that the peptide alone was responsible for the biological activity. Inflammasomes are important for the activation of the innate immune response by detection of danger signals in response to pathogen infection and sterile injury. The inflammasomes modulate the activation of IL-1β and IL-18, by controlling the activation of caspase-1 that cleaves these cytokines for secretion. In fibroblasts, the activation of the inflammasome mediated signaling events by acALY18 was inhibited by Z-YVAD(OMe)-FMK (caspase-1 inactivator) and secretion of IL-1β and IL-18 was abolished. Inflammasome activation in primary human fibroblasts suggests that acALY-18 could be prophylactically useful for the prevention of hospital-acquired infections. Therefore, we conducted a pilot study consisting of two cohorts of 30 mice each infected with MRSA +/- 100 μg/Kg acALY18. In both cohorts, acALY18 reduced the pathogen load in the tissues resulting in smaller lesions and an increased healing rate whereas the untreated mice had much larger lesions with a thick, purulent exudate and no signs of healing. This data suggests that in vivo administration of acALY-18 could induce innate immune responses that would be prophylactically useful for the prevention of hospital-acquired infections.
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