A novel assay of antimycobacterial activity and phagocytosis by human neutrophils

SummaryDespite abundant evidence that neutrophils arrive early at sites of mycobacterial disease and phagocytose organisms, techniques to assay phagocytosis or killing of mycobacteria by these cells are lacking. Existing assays for measuring the antimycobacterial activity of human leukocytes require cell lysis which introduces new bioactive substances and may be incomplete. They are also time-consuming and carry multiple risks of inaccuracy due to serial dilution and organism clumping. Flow cytometric techniques for measuring phagocytosis of mycobacteria by human cells have failed to adequately address the effects of organism clumping, quenching agents and culture conditions on readouts.Here we present a novel in-tube bioluminescence-based assay of antimycobacterial activity by human neutrophils. The assay yields intuitive results, with improving restriction of mycobacterial bioluminescence as the ratio of cells to organisms increases. We show that lysis of human cells is not required to measure luminescence accurately.We also present a phagocytosis assay in which we have minimised the impact of mycobacterial clumping, investigated the effect of various opsonisation techniques and established the correct usage of trypan blue to identify surface-bound organisms without counting dead cells. The same multiplicity of infection and serum conditions are optimal to demonstrate both internalisation and restriction of mycobacterial growth

Hairpin ribozyme-antisense RNA constructs can act as molecular lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5′- and 3′-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-α (TNF-α) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns

Antimicrobial Peptides in Innate Immunity against Mycobacteria

Antimicrobial peptides/proteins are ancient and naturallyoccurring antibiotics in innate immune responses in a variety of organisms. Additionally, these peptides have been recognized as important signaling molecules in regulation of both innate and adaptive immunity. During mycobacterial infection, antimicrobial peptides including cathelicidin, defensin, and hepcidin have antimicrobial activities against mycobacteria, making them promising candidates for future drug development. Additionally, antimicrobial peptides act as immunomodulators in infectious and inflammatory conditions. Multiple crucial functions of cathelicidins in antimycobacterial immune defense have been characterized not only in terms of direct killing of mycobacteria but also as innate immune regulators, i.e., in secretion of cytokines and chemokines, and mediating autophagy activation. Defensin families are also important during mycobacterial infection and contribute to antimycobacterial defense and inhibition of mycobacterial growth both in vitro and in vivo. Hepcidin, although its role in mycobacterial infection has not yet been characterized, exerts antimycobacterial effects in activated macrophages. The present review focuses on recent efforts to elucidate the roles of host defense peptides in innate immunity to mycobacteria

Весовые соотношения тимуса и надпочечников у антенатально погибших плодов

ТИМУСНАДПОЧЕЧНИКИтимомегалиядети первого года жизнидети раннего возрастаМЛАДЕНЕЦ, СМЕРТНОСТЬЭНДОКРИННОЙ СИСТЕМЫ БОЛЕЗНИСМЕРТНОСТЬ ДЕТСКАЯсиндром внезапной детской смерт

Mycobacterium abscessus Glycopeptidolipid Prevents Respiratory Epithelial TLR2 Signaling as Measured by HβD2 Gene Expression and IL-8 Release

Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL) expressing variant (smooth phenotype) in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs) which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8) and human β-defensin 2 (HβD2) are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2) receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with “masking” of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system

Host Antimicrobial Peptides: the promise of new treatment strategies against Tuberculosis

Tuberculosis (TB) continues to be a devastating infectious disease and remerges as a global health emergency due to an alarming rise of antimicrobial resistance to its treatment. Despite of the serious effort that has been applied to develop effective antitubercular chemotherapies, the potential of antimicrobial peptides (AMPs) remains underexploited. A large amount of literature is now accessible on the AMP mechanisms of action against a diversity of pathogens; nevertheless, research on their activity on mycobacteria is still scarce. In particular, there is an urgent need to integrate all available interdisciplinary strategies to eradicate extensively drug-resistant Mycobacterium tuberculosis strains. In this context, we should not underestimate our endogenous antimicrobial proteins and peptides as ancient players of the human host defense system. We are confident that novel antibiotics based on human AMPs displaying a rapid and multifaceted mechanism, with reduced toxicity, should significantly contribute to reverse the tide of antimycobacterial drug resistance. In this review, we have provided an up to date perspective of the current research on AMPs to be applied in the fight against TB. A better understanding on the mechanisms of action of human endogenous peptides should ensure the basis for the best guided design of novel antitubercular chemotherapeutics

Staphylococcus aureus Keratinocyte Invasion Is Dependent upon Multiple High-Affinity Fibronectin-Binding Repeats within FnBPA

Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs) to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs) which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α5β1 integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity) FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA