Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage.

Neuroinflammation has been proven to exert an important effect on brain injury after intracerebral hemorrhage (ICH). Previous studies reported that Didymin possessed anti-inflammatory properties after acute hepatic injury, hyperglycemia-induced endothelial dysfunction, and death. However, the role of Didymin in microglial pyroptosis and neuroinflammation after ICH is unclear. The current study aimed to investigate the effect of Didymin on neuroinflammation mediated by microglial pyroptosis in mouse models of ICH and shed some light on the underlying mechanisms. In this study, we observed that Didymin treatment remarkably improved neurobehavioral performance and decreased BBB disruption and brain water content. Microglial activation and neutrophil infiltration in the peri-hematoma tissue after ICH were strikingly mitigated by Didymin as well. At the molecular level, administration of Didymin significantly unregulated the expression of Rkip and downregulated the expression of pyroptotic molecules and inflammatory cytokines such as Nlrp3 inflammasome, GSDMD, caspase-1, and mature IL-1ß, TNF-α, and MPO after ICH. Besides, Didymin treatment decreased the number of Caspase-1-positive microglia and GSDMD-positive microglia after ICH. Inversely, Locostatin, an Rkip-specific inhibitor, significantly abolished the anti-pyroptosis and anti-neuroinflammation effects of Didymin. Moreover, Rkip binding with Asc could interrupt the activation and assembly of the inflammasome. Mechanistically, inhibition of Caspase-1 by VX-765 attenuated brain injury and suppressed microglial pyroptosis and neuroinflammation by downregulation of GSDMD, mature IL-1ß, TNF-α, and MPO based on Locostatin-treated ICH. Taken together, Didymin alleviated microglial pyroptosis and neuroinflammation, at least in part through the Asc/Caspase-1/GSDMD pathway via upregulating Rkip expression after ICH. Therefore, Didymin may be a potential agent to attenuate neuroinflammation via its anti-pyroptosis effect after ICH.

Trimetazidine affects pyroptosis by targeting GSDMD in myocardial ischemia/reperfusion injury.

OBJECTIVE: Trimetazidine (TMZ) exerts a strong inhibitory effect on ischemia/reperfusion (I/R) injury. Inflammation plays a key role in I/R injury. We hypothesized that TMZ may protect cardiomyocytes from I/R injury by inhibiting inflammation. METHODS: The left anterior descending coronary artery was ligated for 30 min followed by 6 h of reperfusion to establish a model of I/R injury. H9c2 cardiomyocytes were subjected to 2 h of hypoxia and 3 h of normoxic conditions to establish a model of hypoxia/reoxygenation (H/R) injury. We monitored the change in pyroptosis by performing Western blot analysis, microscopy and ELISA. RESULTS: I/R and H/R treatment stimulated gasdermin D-N domain (GSDMD-N) expression in cardiomyocytes (sham onefold vs. I/R 2.5-fold; control onefold vs. H/R 2.0-fold). Moreover, TMZ increased the viability of H9c2 cardiomyocytes subjected to H/R treatment (H/R 65.0% vs. H/R + TMZ 85.3%) and reduced the infarct size in vivo (I/R 47.0% vs. I/R + TMZ 28.3%). H/R and I/R treatment increased the levels of TLR4, MyD88, phospho-NF-κB p65 and the NLRP3 inflammasome; however, TMZ reduced the expression of these proteins. Additionally, TMZ inhibited noncanonical inflammasome signaling induced by I/R injury. CONCLUSIONS: In summary, TMZ alleviated pyroptosis induced by myocardial I/R injury through the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, TMZ represents an alternative treatment for myocardial I/R injury.

Neutrophil Extracellular Traps (NETs) and Covid-19: A new frontiers for therapeutic modality.

Coronavirus disease 2019 (Covid-19) is a worldwide infectious disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). In severe SARS-CoV-2 infection, there is severe inflammatory reactions due to neutrophil recruitments and infiltration in the different organs with the formation of neutrophil extracellular traps (NETs), which involved various complications of SARS-CoV-2 infection. Therefore, the objective of the present review was to explore the potential role of NETs in the pathogenesis of SARS-CoV-2 infection and to identify the targeting drugs against NETs in Covid-19 patients. Different enzyme types are involved in the formation of NETs, such as neutrophil elastase (NE), which degrades nuclear protein and release histones, peptidyl arginine deiminase type 4 (PADA4), which releases chromosomal DNA and gasdermin D, which creates pores in the NTs cell membrane that facilitating expulsion of NT contents. Despite of the beneficial effects of NETs in controlling of invading pathogens, sustained formations of NETs during respiratory viral infections are associated with collateral tissue injury. Excessive development of NETs in SARS-CoV-2 infection is linked with the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) due to creation of the NETs-IL-1ß loop. Also, aberrant NTs activation alone or through NETs formation may augment SARS-CoV-2-induced cytokine storm (CS) and macrophage activation syndrome (MAS) in patients with severe Covid-19. Furthermore, NETs formation in SARS-CoV-2 infection is associated with immuno-thrombosis and the development of ALI/ARDS. Therefore, anti-NETs therapy of natural or synthetic sources may mitigate SARS-CoV-2 infection-induced exaggerated immune response, hyperinflammation, immuno-thrombosis, and other complications.

Wogonin Alleviates Cisplatin-induced Cardiotoxicity in Mice Via Inhibiting Gasdermin D-mediated Pyroptosis.

ABSTRACT: Cardiotoxicity has been well documented as a side effect of cisplatin (CDDP) treatment. The inflammatory response plays a crucial role in the pathological process of CDDP-induced cardiotoxicity. Wogonin is a natural flavonoid compound that possesses cardioprotective and anti-inflammatory qualities. Knowledge of the pharmacological effect and mechanism of wogonin could reveal an efficient way to identify therapeutic strategies. In this study, the potential of wogonin to antagonize CDDP-induced cardiotoxicity was evaluated in C57BL/6 mice in vivo and in H9c2 cells in vitro. The results showed that wogonin protected against CDDP-induced cardiac dysfunction, myocardial injury, and pyroptosis in vivo. Using a Gasdermin D expression plasmid, we revealed that wogonin dramatically reduced CDDP-induced pyroptosis by modulating the Gasdermin D protein in H9c2 cells. In conclusion, wogonin has great potential in attenuating CDDP-induced cardiotoxicity. In addition, greater emphasis should be placed on the antipyroptotic effects of wogonin for the treatment of other diseases.

Pan-cancer analysis reveals the expression, genetic alteration and prognosis of pyroptosis key gene GSDMD.

BACKGROUND: Gasdermins (GSDMs)-mediated pyroptosis is widely involved in activating anti-tumor immunity and suppressing tumor growth. However, whether gasdermin D (GSDMD)-mediated pyroptosis affects patient prognosis in pan-cancer remains unknown. METHODS: We performed analyses of the RNA expression, genetic alteration, prognosis and immune infiltration of GSDMD in pan-cancer. In order to explore the relationship between pyroptosis and tumors, we calculated the correlation between GSDMD and pyroptosis key genes in pan-cancer. We also investigated the enrichment pathway of GSDMD-related genes. RESULTS: GSDMD was differentially expressed in the vast majority of cancer, and could be used as a prognostic marker in adrenocortical carcinoma (ACC), kidney renal clear cell carcinoma (KIRC), brain lower grade glioma (LGG), liver hepatocellular carcinoma (LIHC), skin cutaneous melanoma (SKCM) and rectum adenocarcinoma (READ). Strong evidence indicated the significant correlation of GSDMD with almost all immune checkpoints and immune cells. Pyroptosis-related genes strongly associated with GSDMD in ACC, KIRC, LGG, LIHC and SKCM, suggesting that GSDMD-mediated pyroptosis might play a critical role in the five cancers. CONCLUSION: All the evidence supported the potential role of GSDMD-mediated pyroptosis in cancer. Our results provided new insights into GSDMD as a prognostic marker and potential therapeutic target for cancer.

Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule.

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Structural Basis of the Pore-Forming Toxin/Membrane Interaction.

With the rapid growth of antibiotic-resistant bacteria, it is urgent to develop alternative therapeutic strategies. Pore-forming toxins (PFTs) belong to the largest family of virulence factors of many pathogenic bacteria and constitute the most characterized classes of pore-forming proteins (PFPs). Recent studies revealed the structural basis of several PFTs, both as soluble monomers, and transmembrane oligomers. Upon interacting with host cells, the soluble monomer of bacterial PFTs assembles into transmembrane oligomeric complexes that insert into membranes and affect target cell-membrane permeability, leading to diverse cellular responses and outcomes. Herein we have reviewed the structural basis of pore formation and interaction of PFTs with the host cell membrane, which could add valuable contributions in comprehensive understanding of PFTs and searching for novel therapeutic strategies targeting PFTs and interaction with host receptors in the fight of bacterial antibiotic-resistance.

Luteolin decreases the pathogenicity of Aeromonas hydrophila via inhibiting the activity of aerolysin.

Aeromonas hydrophila (A. hydrophila) can cause a number of diseases in both human and animals. A. hydrophila-related infections in aquaculture cause severe economic losses every year throughout the world. The emergence of antibiotic resistance that is due to the abuse of antibiotics has limited the application of antibiotics. Thus, novel approaches are needed to combat with treatment failure of antibiotics caused by resistant bacterial strains. Aerolysin plays a critical role in the pathogenesis of A. hydrophila and has been considered as a novel target for developing drugs based on anti-virulence strategies. Here, we reported that luteolin, a natural product with no anti-A. hydrophila activity, could reduce aerolysin-induced hemolysis by inhibiting aerolysin activity. The binding mode was simulated by molecular docking and dynamics simulation. Then the main binding sites were confirmed by fluorescence quenching assays. We found that luteolin could hindered the formation of functional heptamer of aerolysin according to the results of the oligomerization assay. Moreover, luteolin could protect A549 cells from aerolysin mediated cell death and increase the survival rate of A. hydrophila-infected channel catfish. These findings suggest a novel approach to developing drugs fighting against A. hydrophila, and luteolin can be a promising drug candidate for treatment of A. hydrophila-associated infections.

Inhibition of Pore-Forming Proteins.

Perforation of cellular membranes by pore-forming proteins can affect cell physiology, tissue integrity, or immune response. Since many pore-forming proteins are toxins or highly potent virulence factors, they represent an attractive target for the development of molecules that neutralize their actions with high efficacy. There has been an assortment of inhibitors developed to specifically obstruct the activity of pore-forming proteins, in addition to vaccination and antibiotics that serve as a plausible treatment for the majority of diseases caused by bacterial infections. Here we review a wide range of potential inhibitors that can specifically and effectively block the activity of pore-forming proteins, from small molecules to more specific macromolecular systems, such as synthetic nanoparticles, antibodies, antibody mimetics, polyvalent inhibitors, and dominant negative mutants. We discuss their mechanism of inhibition, as well as advantages and disadvantages.

Pharmacological Targeting of Pore-Forming Toxins as Adjunctive Therapy for Invasive Bacterial Infection.

For many of the most important human bacterial infections, invasive disease severity is fueled by the cell damaging and pro-inflammatory effects of secreted pore-forming toxins (PFTs). Isogenic PFT-knockout mutants, e.g., Staphylococcus aureus lacking α-toxin or Streptococcus pneumoniae deficient in pneumolysin, show attenuation in animal infection models. This knowledge has inspired multi-model investigations of strategies to neutralize PFTs or counteract their toxicity as a novel pharmacological approach to ameliorate disease pathogenesis in clinical disease. Promising examples of small molecule, antibody or nanotherapeutic drug candidates that directly bind and neutralize PFTs, block their oligomerization or membrane receptor interactions, plug establishment membrane pores, or boost host cell resiliency to withstand PFT action have emerged. The present review highlights these new concepts, with a special focus on ß-PFTs produced by leading invasive human Gram-positive bacterial pathogens. Such anti-virulence therapies could be applied as an adjunctive therapy to antibiotic-sensitive and -resistant strains alike, and further could be free of deleterious effects that deplete the normal microflora.

Perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis.

CD8 T cells protect the liver against viral infection, but can also cause severe liver damage that may even lead to organ failure. Given the lack of mechanistic insights and specific treatment options in patients with acute fulminant hepatitis, we develop a mouse model reflecting a severe acute virus-induced CD8 T cell-mediated hepatitis. Here we show that antigen-specific CD8 T cells induce liver damage in a perforin-dependent manner, yet liver failure is not caused by effector responses targeting virus-infected hepatocytes alone. Additionally, CD8 T cell mediated elimination of cross-presenting liver sinusoidal endothelial cells causes endothelial damage that leads to a dramatically impaired sinusoidal perfusion and indirectly to hepatocyte death. With the identification of perforin-mediated killing as a critical pathophysiologic mechanism of liver failure and the protective function of a new class of perforin inhibitor, our study opens new potential therapeutic angles for fulminant viral hepatitis.

Magnolol protects channel catfish from Aeromonas hydrophila infection via inhibiting the expression of aerolysin.

Aeromonas hydrophila is a common zoonotic pathogen which can cause several infections both in human and animals, particular aquatic animals. Antibiotics have been widely used in the treatment of A. hydrophila infections, however, the development of resistance has limited the treatment for these infections. There is an urgent need for novel agents and strategies against these infections. Aerolysin, a pore-forming toxin secreted by most pathogenic A. hydrophila, is known to contribute to the pathogenesis of A. hydrophila infections. Therefore, aerolysin has been identified as a potential target for drug discovery. In this paper, we found that magnolol, a natural compound without anti -A. hydrophila activity, could significantly inhibit the hemolytic activity of A. hydrophila culture supernatants by inhibiting the transcription of the aerolysin encoding gene aerA at low concentrations. Furthermore, the survival assay showed that magnolol could significantly reduce the mortality induced by A. hydrophila infection in channel catfish (Ictalurus punctatus). Taken together, these findings provide a potent agent against A. hydrophila infections.

The prophylactic effects of human IgG derived from sera containing high anti-PcrV titers against pneumonia-causing Pseudomonas aeruginosa.

The PcrV cap structure of the type III secretory apparatus of Pseudomonas aeruginosa is a vaccine target. Human immunoglobulin G (IgG) molecules extracted from sera containing high or low anti-PcrV titers were tested for their effects against P. aeruginosa pneumonia in a mouse model. Among 198 volunteers, we selected the top 10 high anti-PcrV titer sera and the bottom 10 low anti-PcrV titer sera and extracted the IgG fraction from each serum sample. First, we examined the effects of the IgG against virulent P. aeruginosa. A lethal dose of P. aeruginosa premixed with saline, low titer human IgG, high titer human IgG, or rabbit-derived polyclonal anti-PcrV IgG was intratracheally administered into the lungs of mice, and their survival and lung inflammation were evaluated for 24 h. The high anti-PcrV titer human IgG had a prophylactic effect. Next, the prophylactic effects of intravenous administration of extracted and pooled high or low anti-PcrV titer human IgG were examined. Here, prophylactic intravenous administration of pooled high anti-PcrV titer human IgG, which showed binding capacity to P. aeruginosa PcrV, was more effective than the administration of its low titer pooled equivalent, and the measured physiological and inflammatory parameters correlated with the anti-PcrV titer levels. This result indirectly implies that high anti-PcrV titers in blood can help to protect against virulent P. aeruginosa infections. In addition, the IgG fractions from such high titer sera have potential to be a source of specific intravenous immunoglobulin products for passive vaccination against virulent P. aeruginosa infections.

Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2.

Perforin-2 (MPEG1) is an effector of the innate immune system that limits the proliferation and spread of medically relevant Gram-negative, -positive, and acid fast bacteria. We show here that a cullin-RING E3 ubiquitin ligase (CRL) complex containing cullin-1 and ßTrCP monoubiquitylates Perforin-2 in response to pathogen associated molecular patterns such as LPS. Ubiquitylation triggers a rapid redistribution of Perforin-2 and is essential for its bactericidal activity. Enteric pathogens such as Yersinia pseudotuberculosis and enteropathogenic Escherichia coli disarm host cells by injecting cell cycle inhibiting factors (Cifs) into mammalian cells to deamidate the ubiquitin-like protein NEDD8. Because CRL activity is dependent upon NEDD8, Cif blocks ubiquitin dependent trafficking of Perforin-2 and thus, its bactericidal activity. Collectively, these studies further underscore the biological significance of Perforin-2 and elucidate critical molecular events that culminate in Perforin-2-dependent killing of both intracellular and extracellular, cell-adherent bacteria.

High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore.

Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins (1) provide a window on single molecule interaction with proteins in real time, (2) report on the behavior of macromolecules in confinement, and (3) enable label-free single molecule sensing. Using pores formed by the staphylococcal toxin α-hemolysin (aHL), a particularly pertinent observation was that, under high salt conditions (3-4 M KCl), the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by poly(ethylene glycol) (PEG) oligomers (degree of polymerization approximately 10-60). Notably, this block showed monomeric sensitivity on the degree of polymerization of individual oligomers, allowing the construction of size or mass spectra from the residual current values. Here, we show that the current through the pore formed by aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with drastic differences in the voltage-dependence of the interaction. In contrast to aHL, AeL strongly binds PEG at high transmembrane voltages. This fact, which is likely related to AeL's highly charged pore wall, allows discrimination of polymer sizes with particularly high resolution. Multiple applications are now conceivable with this pore to screen various nonionic or charged polymers.

Direct neutralization of type III effector translocation by the variable region of a monoclonal antibody to Yersinia pestis LcrV.

Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersinia outer proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab')2, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.

Exploration of a series of 5-arylidene-2-thioxoimidazolidin-4-ones as inhibitors of the cytolytic protein perforin.

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure-activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 µM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed T1/2 values of 1.1-1.2 h (dose of 5 mg/kg i.v.) and MTDs of 60-80 mg/kg (i.p.).

Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.

Intimate cell conjugate formation and exchange of membrane lipids precede apoptosis induction in target cells during antibody-dependent, granulocyte-mediated cytotoxicity.

Ab-dependent polymorphonuclear granulocyte (PMN)-mediated cytotoxicity may play an important role in the control of malignant diseases. However, little is known as to which particular pathways are used for the killing of malignant cells by PMN. The production of reactive oxygen intermediates (ROI) has been observed to occur during Ab-dependent, cell-mediated cytotoxicity (ADCC). However, PMN from a patient with chronic granulomatous disease demonstrated strong ADCC against malignant lymphoma cells. Furthermore, the inhibition of ROI production in PMN from healthy donors had no significant effect on ADCC. Therefore, ROI production by the NADPH oxidase of PMN does not appear to be mandatory for PMN-mediated ADCC. Recent data suggest a role for perforins in PMN-mediated cytotoxicity. However, in our assays concanamycin A, an inhibitor of perforin-mediated ADCC by mononuclear cells, had no inhibitory effect on PMN-mediated ADCC. Using electron microscopy we observed that PMN and their target cells intimately interact with the formation of interdigitating membrane protrusions. During PMN and target cell contact there was a mutual exchange of fluorescent membrane lipid dyes that was strongly increased in the presence of tumor-targeting Abs. This observation may be closely related to the recently described process of trogocytosis by lymphocytes. The presence of transient PMN-tumor cell aggregates and the accumulation of PMN with tumor cell-derived membrane lipids and vice versa were associated with effective ADCC as measured by chromium-release or apoptosis induction.

Pore-forming toxins and cellular non-immune defenses (CNIDs).

Pore-forming toxins (PFTs) are the most common class of bacterial protein toxin and are important for bacterial pathogenesis. Recent studies have shown that the previous model stating that epithelial cells lyse in response to these toxins and have no defenses against these pores is oversimplified. Rather, it appears that cells have sophisticated mechanisms and signal-transduction pathways with which to respond to such an attack. There is a growing body of knowledge about how cells respond to and protect themselves against PFTs; this protection against PFTs is likely to be important in host survival to attack by bacterial pathogens, but does not neatly fit into current concepts of adaptive or innate immunity. Therefore, it is proposed that the terminology cellular non-immune defenses (CNIDs) be used to describe defenses that are employed by non-immune cells to protect against bacterial attack.