There is a strong need for a new broad-spectrum antiinfluenza therapeutic, as vaccination and existing treatments are only moderately effective. We previously engineered a lectin, H84T banana lectin ...(H84T), to retain broad-spectrum activity against multiple influenza strains, including pandemic and avian, while largely eliminating the potentially harmful mitogenicity of the parent compound. The amino acid mutation at position 84 from histidine to threonine minimizes the mitogenicity of the wild-type lectin while maintaining antiinfluenza activity in vitro. We now report that in a lethal mouse model H84T is indeed nonmitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus–endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a broad-spectrum antiinfluenza agent.
Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation ...of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The lung epithelium is dynamic, capable of considerable structural and functional plasticity in response to pathogen challenges. Our laboratory has demonstrated that an inhaled combination of a ...Toll-like receptor (TLR) 2/6 agonist and a TLR9 agonist (Pam2ODN) results in robust protection against otherwise lethal pneumonias. We have previously shown that intact epithelial TLR signaling and generation of multisource epithelial reactive oxygen species (ROS) are required for inducible protection. Further investigating the mechanisms underlying this phenomenon of inducible resistance, reverse-phase protein array analysis demonstrated robust STAT3 (signal transducer and activator of transcription 3) phosphorylation following treatment of lung epithelial cells. We show here that Pam2ODN-induced STAT3 phosphorylation is IL-6-independent. We further found that therapeutic epithelial STAT3 activation is required for inducible protection against
pneumonia. Additional studies showed that inhibiting epithelial dual oxidases or scavenging ROS significantly reduced the Pam2ODN induction of STAT3 phosphorylation, suggesting a proximal role for ROS in inducible STAT3 activation. Dissecting these mechanisms, we analyzed the contributions of redox-sensitive kinases and found that Pam2ODN activated epithelial growth factor receptor in an ROS-dependent manner that is required for therapeutically inducible STAT3 activation. Taken together, we demonstrate that epithelial STAT3 is imperative for Pam2ODN's function and describe a novel redox-based mechanism for its activation. These key mechanistic insights may facilitate strategies to leverage inducible epithelial resistance to protect susceptible patients during periods of peak vulnerability.
This study investigates the machining of FLC-4608 (designation by Metal Powder Industries Federation, standard 35) sinter-hardened steel compacts with 90% relative density during turning operation. ...The objective of the study is to analyze the effect of cutting velocity and feed rate on the cutting force component in the direction of cutting motion using chip characteristics. The results showed that the combination of high cutting velocity and low feed rate is the appropriate condition to obtain a low value of the cutting force component. The results also indicated that the machining configurations considered produce shear-localized segmented chips, also known as saw tooth chips, and that the chip formation process involves almost complete densification of the uncut chip material. Except for chip length, all the investigated chip characteristics, minimum and maximum chip thickness, shear band microstructure, and structure below the tip of the chip segment were consistent with the results of the cutting force component. As the feed rate increased, the minimum and maximum chip thickness increased, which was consistent with the increasing value of the cutting force component. Similarly, through the microstructure of the adiabatic shear band and the structure below the tip of the chip segment, increasing cutting velocity showed the dominance of the thermal softening effect over strain hardening and strain rate hardening, consistent with the decreasing value of the cutting force component. This approach is novel, as chip characteristics have received little attention in previous studies on the machining of PM materials. The present study is potentially helpful to the PM industry in achieving better machining process control through a thorough understanding of the results related to the cutting force component in the direction of the cutting motion. The future scope discussed in this report also has prospects for advancing the science of machining PM materials.
Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in ...K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection. Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis.
Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs’ intrinsic host defenses by therapeutic delivery of ...certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (Δ
Ψm
), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness, and airway remodeling. Epidemiologic ...data reveal that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens
expression of myriad Toll-like receptors (TLRs) and other pattern-recognition receptors. We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 ("Pam2", TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 ("ODN", TLR9 ligand), when delivered together by aerosol ("Pam2ODN"), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of the lung, and reduces the likelihood of inappropriate sensitization to aeroallergens. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy.
Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of ...synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.
Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.
Despite widespread infection prevention efforts, pneumonia remains the leading cause of death among patients with acute leukemia, due to complex disease- and treatment-dependent immune defects. We ...have reported that a single inhaled treatment with a synergistic combination of Toll-like receptor 2/6 (TLR 2/6) and TLR9 agonists (Pam2-ODN) induces protective mucosal defenses in mice against a broad range of pathogens. As Pam2-ODN–induced protection persists despite depletion of several leukocyte populations, we tested whether it could prevent pneumonia in a mouse model of acute myeloid leukemia (AML) remission induction therapy. Pam2-ODN prevented death due to pneumonia caused by Pseudomonas aeruginosa, Streptococcus pneumoniae, and Aspergillus fumigatus when mice were heavily engrafted with leukemia cells, had severe chemotherapy-induced neutropenia or both. Pam2-ODN also extended survival of pneumonia in NSG mice engrafted with primary human AML cells. Protection was associated with rapid pathogen killing in the lungs at the time of infection and with reduced pathogen burdens at distant sites at the end of observation. Pathogen killing was inducible directly from isolated lung epithelial cells and was not abrogated by the presence of leukemia cells or cytotoxic agents. Pam2-ODN had no discernible effect on replication rate, total tumor population, or killing by chemotherapy of mouse or human leukemia cells, either in vitro or in vivo. Taken together, we report that therapeutic stimulation of lung epithelial defenses robustly protects against otherwise lethal pneumonias despite the profound immune dysfunction associated with acute leukemia and its treatment. These findings may suggest an opportunity to protect this population during periods of peak vulnerability.
•Survival of acute myelogenous leukemia is frequently limited by pneumonia, due to disease- and therapy-associated immune defects.•Inducible epithelial resistance protects neutropenic, leukemic mice against lethal pneumonia without impacting AML cell proliferation.
Though powder metallurgy route of product fabrication receives recognition for near-net-shape abilities, economic constraints often demand involvement of minimal machining in the process. To overcome ...difficulties associated with machining of sintered/sinter-hardened steels, concept of machining their un-sintered (green) and partially sintered (pre-sintered) counter-parts have been explored since last twenty-five to thirty years. ‘Green machining’ defines machining of green state powder compacts, and this communication describes the first report that reviews green machining of powder metallurgy steels. Schematic and theoretical models have been introduced in this review, to illustrate the phenomenal consequences commonly observed during green machining practices. The report focuses on the previous attempts on green machinability improvements, which are broadly classified herewith as - I through dealing with strength of the green compact, and II through dealing with the cutting conditions. Besides this, the description considers aggregate studies on green machining and machining post sintering/sinter-hardening. Such studies not only discuss analogies and differences between the machining avenues, but also signify effects of green machining on mechanical behavior of sintered/sinter-hardened steels. Further, the report briefly introduces machining of pre-sintered compacts, as well. As and when entailed, the issues involved in green machining are discussed and future scope for advancements are suggested.
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