No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Oxidative burst without phagocytes: The role of respiratory proteins
Abstract
Respiratory proteins of vertebrates (hemoglobin) and invertebrates (hemocyanin) can generate highly reactive oxygen intermediates. New work shows that microbial compounds directly activate this release of reactive oxygen intermediates that kills the microbes.
... Systems exist to detoxify Hb (14), but Hb is also an innate immune factor with evolutionarily-conserved antimicrobial function. When extracellular Hb is activated by proteolytic cleavage, bacterial binding, or conformational change, it increases production of ROS (reactive oxygen species) (15); this process has been called "oxidative burst without phagocytes" (15,16). Our earlier work used evolutionary conservation to identify ROS-producing fragments of Hb and crosstalk with tissue factor coagulation (17,18). ...
... Systems exist to detoxify Hb (14), but Hb is also an innate immune factor with evolutionarily-conserved antimicrobial function. When extracellular Hb is activated by proteolytic cleavage, bacterial binding, or conformational change, it increases production of ROS (reactive oxygen species) (15); this process has been called "oxidative burst without phagocytes" (15,16). Our earlier work used evolutionary conservation to identify ROS-producing fragments of Hb and crosstalk with tissue factor coagulation (17,18). ...
... Nevertheless, some pathogens (e.g., hemolytic streptococci) can liberate iron faster than the host can sequester it. In response, the innate immune system has mechanisms for amplifying ROS production by hemglobin in settings of hemolysis (15)(16)(17)(18) rather minimizing the cytotoxicity. Some of this may be relevant to the toxicity of myolysis in mPI. ...
The reasons for poor healing of pressure injuries are poorly understood. Vascular ulcers are worsened by extracellular release of hemoglobin, so we examined the impact of myoglobin (Mb) iron in murine muscle pressure injuries (mPI). Tests used Mb-knockout or treatment with deferoxamine iron chelator (DFO).
Unlike acute injuries (from cardiotoxin), mPI regenerated poorly with a lack of viable immune cells, failure of phagocytosis, and abnormal deposition of iron. However, Mb-knockout or DFO-treated mPI displayed a reversal of the pathology: decreased tissue death, decreased iron deposition, decrease in markers of oxidative damage, and higher numbers of intact immune cells. Subsequently, DFO treatment improved myofiber regeneration and morphology.
We conclude that myoglobin iron contributes to tissue death in mPI. Remarkably, a large fraction of muscle death in untreated mPI occurred later than, and was preventable by, DFO treatment, even though treatment started 12 hours after pressure was removed. This demonstrates an opportunity for post-pressure prevention to salvage tissue viability.
... Exposure of Hc isolated from the hemolymph of the Mangrove horseshoe crab, Carcinoscorpius rotundicauda, to a range of microbial proteases and ligands in vitro, resulted in the production of reactive oxygen species (ROS) and the simultaneous induction of Hc-d PO activity (Jiang et al., 2007). As Hc-d PO catalyses the initial step(s) in melanin biosynthesis, quinone derivatives can enter enzymatic/non-enzymatic redox cycling pathways with their respective semi-quinone counterparts (Fig. 5), culminating in the formation of cytotoxic ROS (Komarov et al., 2005;Bogdan, 2007). Komarov et al. (2005) demonstrated clearly that hydrogen peroxide (H 2 O 2 ) and superoxide O À 2 À Á radicals are produced in abundance during the oxidation of diphenols by mushroom PO. ...
... The lectin-carbohydrate interaction is thought to be mediated by carboxyl and N-acetylated groups of sugars, such as N-acetyl-neuraminic acid. The generation of ROS (and nitrogen species) is an influential component of the invertebrate immune response (Bogdan, 2007) and has been used successfully as an indicator of immune competence in cultured shrimp (Yon-Chin et al., 2008). ...
... AMPs, antimicrobial peptides, PAMPs, pathogen associated molecular patterns, ROS, reactive oxygen species. (Information was obtained from Bolton et al., 2000;Sugumaran, 2002;Fang, 2004;Komarov et al., 2005;Bogdan, 2007;Jiang et al., 2007.) ...
... Th2 cytokines induce arginase 1, which degrades arginine in polyamines and proline that are used for cellular proliferation and collagen production. In contrast, one of the hallmarks of M1 activation is the generation of nitric oxide (NO) by an inducible nitric oxide synthase (NOS2) [8]. ...
... Given that NO plays a major role in killing L. major [8], we examined the expression of Nos2 in C57Bl/6 and BALB-C macrophages after activation with IFN-γ or after addition of lipopolysaccharide (LPS). No differences were found in the expression of Nos2 ( Figure 1D) or its protein (data not shown). ...
... To evade immune responses, some pathogens generate their own arginases [10,27,28] or induce arginase expression in the host [29,30]. Arginine then becomes limited for the production of NO by NOS2, an essential mechanism for host defense against many pathogens [8]. Production of a high level of arginase 1 blocks the immune response locally at the site of pathology, causing local depletion of arginine, which impairs the capacity of T cells in the lesion to proliferate and produce IFN-γ [6]. ...
Host genetic factors play a crucial role in immune response. To determine whether the differences between C57Bl/6 and BALB-C
mice are due only to the production of cytokines by T-helper 1 cells or T-helper 2 cells, we obtained bone marrow–derived
macrophages from both strains and incubated them with these cytokines. Although the induction of Nos2 and Arg1 was similar in the 2 strains, infectivity to Leishmania major differed, as did macrophage uptake of arginine, which was higher in BALB-C macrophages. The levels of interferon γ– and interleukin
4–dependent induction of the cationic amino acid transporter SLC7A2 (also known as “cationic amino acid transporter 2,” or “CAT2”) were decreased in macrophages from C57Bl/6 mice. This reduction
was a result of a deletion in the promoter of one of the 4 AGGG repeats. These results demonstrate that the availability of
arginine controls critical aspects of macrophage activation and reveal a factor for susceptibility to Leishmania infection.
... Recent studies have demonstrated that Hb exhibits "pseudoperoxidase" or peroxidaselike activity in bacteria [16], invertebrates [4,17], and humans [18][19][20][21]. It has been suggested that human Hb may exert antibacterial effects through its peroxidase activity, which catalyzes the production of reactive oxygen species (ROS) [12,19]. ...
... Recent studies have demonstrated that Hb exhibits "pseudoperoxidase" or peroxidaselike activity in bacteria [16], invertebrates [4,17], and humans [18][19][20][21]. It has been suggested that human Hb may exert antibacterial effects through its peroxidase activity, which catalyzes the production of reactive oxygen species (ROS) [12,19]. Moreover, microbial extracellular proteases and pathogen-associated molecular patterns have been shown to enhance Hb's peroxidase activity, promoting the generation of superoxide anions (O 2 − ) [21][22][23][24]. ...
This study investigates the essential role of the heme cavity in the peroxidase and antibacterial activities of homodimeric hemoglobin (Tg-HbI) from the blood clam Tegillarca granosa. After treatment with sodium dodecyl sulfate (SDS), the peroxidase and antibacterial activities of the Tg-HbI were significantly inhibited, with the degree of inhibition correlating positively with the SDS concentration. Fluorescence spectroscopy, UV-Vis spectroscopy, and molecular docking analysis further revealed that SDS interacts with key amino acid residues (e.g., His70 and His102) in the heme cavity of Tg-HbI, causing conformational changes that disrupt the internal hydrophobic interactions, thus inhibiting its function. This study confirms that the antibacterial effect of Tg-HbI is mediated through its peroxidase activity and that the heme cavity plays a critical role in maintaining this activity. These findings lay a foundation for further research on the immune defense functions of hemoglobin and provide new insights into the mechanisms of environmental adaptation in T. granosa.
... Systems exist to detoxify Hb (Cherayil, 2011), but Hb is also an innate immune factor with evolutionarily conserved antimicrobial function. When extracellular Hb is activated by proteolytic cleavage, bacterial binding, or conformational change, it increases production of reactive oxygen species (ROS) Bogdan, 2007; this process has been called 'oxidative burst without phagocytes' (Bogdan, 2007;Jiang et al., 2007). Our earlier work used evolutionary conservation to identify ROS-producing fragments of Hb and crosstalk with tissue factor coagulation (Bahl et al., 2014;Bahl et al., 2011). ...
... Systems exist to detoxify Hb (Cherayil, 2011), but Hb is also an innate immune factor with evolutionarily conserved antimicrobial function. When extracellular Hb is activated by proteolytic cleavage, bacterial binding, or conformational change, it increases production of reactive oxygen species (ROS) Bogdan, 2007; this process has been called 'oxidative burst without phagocytes' (Bogdan, 2007;Jiang et al., 2007). Our earlier work used evolutionary conservation to identify ROS-producing fragments of Hb and crosstalk with tissue factor coagulation (Bahl et al., 2014;Bahl et al., 2011). ...
The reasons for poor healing of pressure injuries are poorly understood. Vascular ulcers are worsened by extracellular release of hemoglobin, so we examined the impact of myoglobin (Mb) iron in murine muscle pressure injuries (mPI). Tests used Mb-knockout or treatment with deferoxamine iron chelator (DFO). Unlike acute injuries from cardiotoxin, mPI regenerated poorly with a lack of viable immune cells, persistence of dead tissue (necro-slough), and abnormal deposition of iron. However, Mb-knockout or DFO-treated mPI displayed a reversal of the pathology: decreased tissue death, decreased iron deposition, decrease in markers of oxidative damage, and higher numbers of intact immune cells. Subsequently, DFO treatment improved myofiber regeneration and morphology. We conclude that myoglobin iron contributes to tissue death in mPI. Remarkably, a large fraction of muscle death in untreated mPI occurred later than, and was preventable by, DFO treatment, even though treatment started 12 hr after pressure was removed. This demonstrates an opportunity for post-pressure prevention to salvage tissue viability.
... 23 Inflammatory markers such as tumour necrosis factor-a and interleukin-6 can disrupt erythropoiesis by direct myelosuppression of erythroid precursors, lowering iron bioavailability for haemoglobin synthesis, promoting apoptosis, increasing erythropoietin resistance in precursor cell lines, and lowering erythropoietin production from the kidney. 24,25 Erythrolysis, on the other hand, is linked to an increase in the production of free radicals, 26 which are known to be detrimental to the heart. Higher oxidative stress is another potential mechanism linking RDW with mortality because it reduces RBC survival and leads to anisocytosis resulting from an increase in circulating premature erythrocytes. ...
Aims:
There is limited evidence for the correlation between short-term mortality and red cell distribution width (RDW) in critical patients with heart failure. Herein, a retrospective cohort study was conducted to investigate whether RDW was independently associated with short-term mortality in critically ill patients with heart failure.
Methods and results:
As a retrospective cohort study, it involved a total of 9465 patients with heart failure from the MIMIC-IV database. The target-dependent and independent variables were in-hospital mortality, 90 day mortality and RDW measured at baseline, respectively. The relationship between all-cause death and baseline RDW in hospital and after 90 days of admission to ICU was evaluated by using the Kaplan-Meier plot and Cox proportional hazard analysis. The average age of participants was 74.4 (64.2, 83.5) years old, among whom about 54.6% were male. Results of the adjusted Cox proportional hazard model revealed that RDW had a positive association with both in-hospital and 90 day mortality risk after the adjustment of confounders (HR = 1.09, 95% CI: 1.04-1.15, P < 0.001; HR = 1.11, 95% CI: 1.08-1.14, P < 0.001, respectively). A non-linear relationship was found between RDW and 90 day mortality, which had a threshold of 14.96%. The effect sizes and confidence intervals below and above the threshold were 1.36 (1.14 to 1.62) and 1.09 (1.04 to 1.15), respectively. It was also found by subgroup analysis that there were stronger correlations in male and patients with normal renal function.
Conclusions:
Our data suggest that the short-term mortality of critically ill patients with HF is independently predicted by RDW. At the same time, large prospective research and longer follow-up time are required to further validate the findings of this study.
... ProPO system is an important component of innate immune response in invertebrate involving in melanization, cytotoxic reactions, cell adhesion encapsulation, phagocytosis and self/non-self recognition (Cerenius et al., 2008;Wang et al., 2018;Bouallegui, 2019). Tyrosinase as one of type of phenoloxidases, a key member of proPO cascades of bivalves was one of the important pathways to product reactive oxygen intermediates (ROIs) to against pathogen invasion, regulation wound healing as signal molecular (Bogdan, 2007;Allam and Raftos, 2015;Wang et al., 2018). Meanwhile, researches also showed that excessive respiratory burst caused by ROIs after nuclear transplantation operation affected the pearl sac development of on pearl mussel of P. fucata (Kishore and Southgate, 2015a), suggesting the importance of oxidation-reduction process in the stress response of pearl oysters to nucleus implantation (Wei et al., 2017a;Wang et al., 2019). ...
Immune response plays an important role in the pearl sac formation and pearl quality. However, there is little knowledge about the mode and mechanism of the immune responses in the pearl sac formation of the pearl mussel Hyriopsis cumingii. In this study, we monitored the process of pearl sac formation by histology examination for 21 days
after grafting. The results showed that a large number of hemocytes aggregated in the pearl sac cavity on day 8 after grafting and provided organic substrate for the deposition of extracellular matrix and CaCO3 crystal and mediated the initiation of pearl forming on day 12 after grafting. These results revealed the important role of hemocytes on
the early pearl sac and pearl formations. In the transcriptome analysis of pearl sac at the key time-points of day 0 and days 6, 8, 12 after grafting, the expression profiles of the immune-related genes revealed the immune response pattern of H. cumingii after grafting. The ignificant up-regulation in the hydrolytic enzymes, acute phase
proteins, and antimicrobial peptides of humoral defense factors mRNA expression on day 6 after grafting suggested that these immune-related gene products were the main immune defense during the early wound healing; Meanwhile, the continuous mRNA
up-regulation of antioxidant enzymes such as Cu-Zn SOD, Se-GPX, and tyrosinase in prophenoloxidase (proPO) system on days 8 and 12 suggested the important role of these immune effectors in oxidation-reduction on the later stage of pearl sac and pearl formations in H. cumingii. Moreover, the results of KEGG analysis of differentially
expressed genes suggested the potential regulatory function of the signaling pathways of proPO system and complement system during pearl sac formation. These results provided valuable new insights into the roles and functions of the immune system in pearl sac and pearl formation.
... ROS are also produced in mammals as normal metabolic species and can provide either benefits or drawbacks, depending on their concentration. Mitochondria [11], the family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) [12], and oxidative burst from respiratory proteins such as hemoglobin [13], are the production sites for ROS in mammals. For example, ROS are produced via a one-electron reduction of O2 in mitochondria to generate water, in which a small amount of O • can be produced as a result of the reduction process. ...
Reactive oxygen species (ROS) have been found in plants, mammals, and natural environmental processes. The presence of ROS in mammals has been linked to the development of severe diseases, such as diabetes, cancer, tumors, and several neurodegenerative conditions. The most common ROS involved in human health are superoxide (O2•−), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH). Organic and inorganic molecules have been integrated with various methods to detect and monitor ROS for understanding the effect of their presence and concentration on diseases caused by oxidative stress. Among several techniques, fluorescence and electrochemical methods have been recently developed and employed for the detection of ROS. This literature review intends to critically discuss the development of these techniques to date, as well as their application for in vitro and in vivo ROS detection regarding free-radical-related diseases. Moreover, important insights into and further steps for using fluorescence and electrochemical methods in the detection of ROS are presented.
... ProPO system is an important component of innate immune response in invertebrate involving in melanization, cytotoxic reactions, cell adhesion encapsulation, phagocytosis and self/non-self recognition (Cerenius et al., 2008;Wang et al., 2018;Bouallegui, 2019). Tyrosinase as one of type of phenoloxidases, a key member of proPO cascades of bivalves was one of the important pathways to product reactive oxygen intermediates (ROIs) to against pathogen invasion, regulation wound healing as signal molecular (Bogdan, 2007;Allam and Raftos, 2015;Wang et al., 2018). Meanwhile, researches also showed that excessive respiratory burst caused by ROIs after nuclear transplantation operation affected the pearl sac development of on pearl mussel of P. fucata (Kishore and Southgate, 2015a), suggesting the importance of oxidation-reduction process in the stress response of pearl oysters to nucleus implantation (Wei et al., 2017a;Wang et al., 2019). ...
Immune response plays an important role in the pearl sac formation and pearl quality. However, there is little knowledge about the mode and mechanism of the immune responses in the pearl sac formation of the pearl mussel Hyriopsis cumingii. In this study, we monitored the process of pearl sac formation by histology examination for 21 days after grafting. The results showed that a large number of hemocytes aggregated in the pearl sac cavity on day 8 after grafting and provided organic substrate for the deposition of extracellular matrix and CaCO3 crystal and mediated the initiation of pearl forming on day 12 after grafting. These results revealed the important role of hemocytes on the early pearl sac and pearl formations. In the transcriptome analysis of pearl sac at the key time-points of day 0 and days 6, 8, 12 after grafting, the expression profiles of the immune-related genes revealed the immune response pattern of H. cumingii after grafting. The significant up-regulation in the hydrolytic enzymes, acute phase proteins, and antimicrobial peptides of humoral defense factors mRNA expression on day 6 after grafting suggested that these immune-related gene products were the main immune defense during the early wound healing; Meanwhile, the continuous mRNA up-regulation of antioxidant enzymes such as Cu-Zn SOD, Se-GPX, and tyrosinase in prophenoloxidase (proPO) system on days 8 and 12 suggested the important role of these immune effectors in oxidation-reduction on the later stage of pearl sac and pearl formations in H. cumingii. Moreover, the results of KEGG analysis of differentially expressed genes suggested the potential regulatory function of the signaling pathways of proPO system and complement system during pearl sac formation. These results provided valuable new insights into the roles and functions of the immune system in pearl sac and pearl formation.
... The immunocytes are also important sources for many humoral molecules by releasing the pro-phenol oxidase activating system (pro-PO-AS) that regulates coagulation and melanization and delivers molecules needed for opsonization (Jiravanichpaisal et al., 2006;Söderhäll and Cerenius, 1998), production of anti-microbial peptides (Tincu and Taylor, 2004) and other cytotoxic compounds such as reactive oxygen and nitrogen species, and hydrolytic enzymes (e.g. lysozyme) (Bogdan, 2007). Vertebrates, such as fish have beside their humoral and cellular defense systems that involve e.g. ...
Manganese (Mn) is an essential trace metal for all organisms. However, in excess it causes toxic effects but the impact on aquatic environments has so far been highly overlooked. Manganese is abundant both in costal and deep sea sediments and becomes bioavailable (Mn2+) during redox conditions. This is an increasing phenomenon due to eutrophication-induced hypoxia and aggravated through the ongoing climate change. Intracellular accumulation of Mn2+ causes oxidative stress and activates evolutionary conserved pathways inducing apoptosis and cell cycle arrest. Here, studies are compiled on how excess of dissolved Mn suppresses the immune system of various aquatic organisms by adversely affecting both renewal of immunocytes and their functionality, such as phagocytosis and activation of pro-phenoloxidase. These impairments decrease the animal's bacteriostatic capacity, indicating higher susceptibility to infections. Increased distribution of pathogens, which is believed to accompany climate change, requires preserved immune sentinel functions and Mn can be crucial for the outcome of host-pathogen interactions.
... Este pigmento está relacionado con la reacción de proPO. Esta reacción está asociada a la defensa de los hemocitos a través de la generación de intermediarios reactivos (Quinonas, proquinonas, radicales de O 2 · -), los cuales son citotóxicos (Bodgan, 2007). En los sitios de injuria, la melanina es generada por los hemocitos sirviendo como una barrera mecánica que impide el crecimiento del invasor o incluso eliminando al agente invasor (Cerenius y Söderhäll, 2004). ...
Marine invertebrates, like bivalves depend exclusively on their innate immunity system to deal with pathogen challenges. In Atlantic mussels it has been shown that hemocytes present in haemolymph, synthesize a variety of antimicrobial peptides, which are released to haemolymph. In endemic mussels of the Chilean coasts, the innate immune system has not been characterized, therefore, the antimicrobial activity of haemolymph components of C. chorus was evaluated in this work.
Using the classic acid and solid phase extractions to isolate antimicrobial peptides from haemolymph, we detected a powerful antibacterial activity in fractions which present very low and medium hydrophobicity These results indicate that in C. chorus would contain antimicrobial peptides with different features than those described in other mussels.
Using May-Grünwald-Giemsa staining haemocytes population was studied and as well as in the other mythilids, basophylic, granular eosinophylic and hialine, were observed. In addition, hemocytes in animals challenged whith E. coli significant morphology change in haemocytes, including degranulation and appearence of a brown pigment, corresponding to melanin were observed. This brown melanin is synthesized by an ancient defense mechanism called the prophenoloxidase reaction. On the other hand, the most abundant protein of haemolymph of C. chorus has a molecular weight of 75 kDa, contrasting with what was described for other mussels, in which a 37 kDa protein is the predominant. This protein was isolated by Ni-IMAC chromatography, which indicates that it may belong to the Histidine Rich Glycoprotein family (HRG), described in other mussels. The apparent molecular weight for this protein determined by gel filtration chromatography under native conditions was more than 1 MDa. Immunodetection of this protein with anti-key hole limpet and C. concholepas haemocyanin antibodies by Western blot, suggests that this protein may have haeomcyanin features. Interestingly, this putative HRG- haemocyanin from C. chorus exhibits antibacterial activity, suggesting that it may be a component of the innate immunity system.
... Hemoglobin can generate highly reactive oxygen intermediates. Microbial compounds directly activate this release of reactive oxygen intermediates that kills the microbes [79]. Microbial proteases and pathogen-associated molecular patterns provoke rapid production of reactive oxygen species. ...
Human innate immunity operates in two compartments: extravascular (the tissues) and intravascular (the bloodstream). Physical conditions (fluid dynamics) in the compartments are different and, as a result, bactericidal mechanisms and involved cells are different as well. In relatively static media (the tissues, lymph nodes), bacteria are killed by phagocytes; in dynamic media (the bloodstream), bacteria are killed by erythrocytes. In the tissues and lymph nodes, resident macrophages and transmigrated from blood leukocytes (neutrophils and monocytes) recognize, engulf, kill, and digest bacteria; the clearance of the bloodstream from bacteria is performed by oxycytosis: erythrocytes catch bacteria by electric charge attraction and kill them by the oxygen released from oxyhemoglobin. Killed by erythrocytes, bacteria are decomposed and digested in the liver and the spleen. Phagocytosis by leukocytes in the tissues and oxycytosis by erythrocytes in the bloodstream are the main bactericidal mechanisms of human innate immunity.
... ROS analysis of HeLa cells treated with HMC was carried out as previously described [37]. In brief, HeLa cells were planted at a density of 5×10 3 /well in a 96-well plate. ...
Hemocyanin (HMC) has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB) assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI) staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2), aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD), heat shock 60kD protein 1 variant 1 (HSP60), heat shock 70kDa protein 5 precursor (HSP70), heat shock protein 90kDa beta member 1 precursor (HSP90), 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1), were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS), mitochondrial membrane potential (Δψm) and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.
... V případě oxidačního vzplanutí neutrofilních granulocytů (mikrofágů) se jedná o purulentní zánětlivý proces. Ten bývá obvykle iniciován extracelulárními bakteriemi [5,12,14,17]. Je ale popsána i jeho neinfekční příčina u některých pacientů s ischemizací tkáně mozku následkem vasospasmů po atace subarachnoidálního krvácení [9,20,22,23]. Pro purulentní zánět infekční i neinfekční příčiny je typická N granulocytární pleocytóza v cytologickém obraze CSF [1,3]. ...
Objective: Evaluate the significance of combined cytology and energy balance in the cerebrospinal fluid compartment for the detection and characterization of the inflammatory processes in the central nervous system (CNS). Design: Retrospective study. Settings: Department of Clinical Biochemistry, Haematology and Immunology, Hospital Kadaň; Department of Clinical Biochemistry, Masaryk Hospital in Ústí nad Labem; Institute of Clinical Immunology and Alergology, Faculty of Medicine Hradec Králové, Charles Univerzity Prague; Laboratory for Liquorology and Neuroimmunology Topelex, Prague; Department of Neurosurgery, J. E. Purkinje University, Masaryk Hospital in Ústí nad Labem; International Clinical Research Center, Brno; Department of Intensive Care Medicine, Masaryk Hospital in Ústí nad Labem; Department of Clinical Biochemistry, Hospital Most. Material and Methods: We examined 437 cerebrospinal fluid (CSF) specimens in patients with serous inflammations of infectious etiology in the CNS (170), purulent inflammations bacterial etiology in the CNS (121), inflammations with oxidative burst of macrophages of infectious and tumorous etiology in the CNS (25) and in patients without CNS impairment (121). We used the Fisher's F-test and the Student's t-test for the statistical evaluation of the energy balance in the cerebrospinal fluid compartment. We subsequently counted the specificities, sensitivities and diagnostic efficiencies for the combined evaluation of the cytological picture of the CSF and energy balance in the CSF compartment. Results: We found highly statistically significant differences (p<0.001) between the variations and the mean values of coefficients of energy balance in all our groups of patients. We achieved very good results in combined evaluations of cytological pictures and the energy balance in the CSF compartment in the group of patients with serous inflammations in the CNS of infectious etiology (specificity = 100.0 %, sensitivity = 97.1 % and diagnostic efficiency = 98.9 %), in the group of patients with purulent inflammation in the CNS bacterial etiology (specificity = 100.0 %, sensitivity = 96.7 % and diagnostic efficiency = 99.1 %), in the group of patients with oxidative burst of macrophages in the CNS infectious etiology (specificity = 98.6 %, sensitivity = 100.0 % and diagnostic efficiency = 98.6 %) and also in the group of patients with oxidative burst of macrophages in the CNS of tumorous etiology (specificity = 98.8 %, sensitivity = 100.0 % and diagnostic efficiency = 98.9 %). Conclusion: Combined evaluation of the cytological pictures and energy balance in the CSF compartment led to reliable differentiation of the CNS impairment into the serous inflammations, the purulent inflammations and the inflammations with oxidative burst of macrophages in our group of patients. It was subsequently possible to specify the correct pathogens more effectively and to identify tumour infiltration of meninges in some cases.
... The potential mechanism that causes ATa via alteration of RDW levels and Pwd is hitherto unknown. Bogdan showed that increased concentrations of free radicals can accelerate the dissolution of erythrocytes, and the diminished erythrocyte number acts as a trigger for hematopoiesis, thereby causing an increase in RDW levels [19]. Both inflammation and oxidative stress can elevate RDW levels [20,21]. ...
Although red cell distribution width (RDW) and P-wave dispersion (Pwd) are strong independent predictors of atrial tachyarrhythmia (ATa), the association between these two factors in the occurrence of ATA has hitherto not been reported. We retrospectively analyzed the cases of ATa patients who underwent Holter monitoring at our hospital from October 2013 to August 2014. Clinical data including RDW and color doppler ultrasonography data were collected, and Pwd was calculated from synchronous recording of P-wave intervals of 12-lead electrocardiograms. Patients were categorized into three groups in ascending order of RDW values. Between-group comparisons yielded significant differences in Pwd, left ventricular diastolic diameter (LVDD), and left atrial diameter (LAD; P < 0.05). Pearson correlation analysis revealed that the RDW level positively correlated with the Kleiger grade of atrial arrhythmia (r = 0.280, P < 0.001), Pwd (r = 0.148, P = 0.001), and LAD (r = 0.297, P < 0.001); Pwd positively correlated with the Kleiger grade of atrial arrhythmia (r = 0.257, P < 0.001), aortic root diameter (r = 0.143, P < 0.002), and LAD (r = 0.201, P < 0.001). Binary logistic multiple regression analysis with ATa as the dependent variable revealed that Pwd [odds ratio (OR) = 1.024], RDW [OR = 1.215], and aortic root diameter [OR = 1.030] were significant risk factors for ATa occurrence. This is the first study to establish a correlation between RDW and Pwd in the occurrence of ATa; however, further prospective studies using large cohorts are required to validate the correlation.
... This H 2 O 2 can then be converted into HOCl or other toxic products through other systems (i.e. halidemyeloperoxidase system) (25). PLD is a regulator of NADPH oxidase, which is sequentially activated by PA and DAG (24). ...
... The methaemoglobin content in a haemoglobin solution is reported to increase from 1.3 to 2.4% during 47 days of storage in a blood bank [31]. Presumably, this would catalyse ROS production, like MPO does [29,[31][32][33]. A further increase (Fig. 4) of haemoglobin concentration in the assay (20 lg/ ml) reduced the chemiluminescence to background values, in line with our tentative explanation of unexpected findings (see above). ...
The homeostatic control of the redox system (the redoxome) in mammalian cells depends upon a large number of interacting molecules, which tend to buffer the electronegativity of cells against oxidants or reductants. Some of these components kill – at high concentration – microbes and by-stander normal cells, elaborated by professional phagocytes. We examined whether a simple, in vitro chemiluminescence set-up, utilizing redox components from human polymorphonuclear neutrophils (PMN) and red blood cells (RBC), could clarify some unexplained workings of the redoxome. PMN or purified myeloperoxidase (MPO) triggers formation of ROS, quantified by light emission from oxidized luminol. Both PMN and RBC can generate abundant amounts of reactive oxygen species (ROS), necessitating the presence of a high-capacity redoxome to keep the cellular electronegativity within physiological limits.We got proof-of-principle evidence that our assay could assess redox effects, but also demonstrated the intricacies of redox reactions. Simple dose-responses were found, as for the PMN proteins MPO, S100A9 (A9) and S100A8 (A8), and the system also revealed the reducing capacity of vitamin B12 (Cbl) and lutein. However, increased concentrations of oxidants in the assay mixture could decrease the chemiluminescence. Even more remarkable, A9 and NaOCl together stimulated the MPO response, but alone they inhibited MPO chemiluminescence. Biphasic responses were also recorded for some dose-response set-ups and are tentatively explained by a “balance hypothesis” for the redoxome.This article is protected by copyright. All rights reserved.
... Our results suggest that NADPH oxidase might contribute to the development of acute inflammation in the colon epithelium. It is well known that this enzyme is rapidly activated during infection or after stimulation by bacterial endotoxins, when innate immune system activates protective cascades against microbial invaders in phagocytic and non-phagocytic cells [26][27][28][29] . ...
Aim:
To investigate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium (DSS)-induced colitis.
Methods:
Balb/c mice were divided into three groups: 8 mice with acute DSS-induced colitis (3.5% DSS solution; 7 d), 8 mice with chronic DSS-induced colitis (3.5% DSS solution for 5 d + water for 6 d; 4 cycles; total: 44 d) and 12 mice without DSS supplementation as a control group. Primary colonic epithelial cells were isolated using chelation method. The cells were cultivated in the presence of mediators (lipopolysaccharide (LPS), apocynin or diphenyleneiodonium). Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorometrically using Amplex Red. Production of tumour necrosis factor-alpha (TNF-α) by the colonic epithelial cells was analysed by ELISA. Nox1 gene expression was assessed by real-time PCR.
Results:
Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups, whereas decreased viability, increased ROS production, and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls. The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis. Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups.
Conclusion:
Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.
... This activity is characterized by the expression of NO synthase 2 (NOS2) and the biosynthesis and release of proinflammatory cytokines, including TNF-a, IL-1b, and IL-6. NOS2 is required for the production of NO (3). Sustained NO production endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria, or fungi, although it also has deleterious effects on host cells and therefore must be tightly regulated (4,5). ...
LPS induces the expression of NO synthase 2 (nos2) in macrophages. The expression of this molecule is one of the hallmarks of classical activation. In this paper, we describe that trichostatin A (TSA), which inhibits deacetylase activity, blocks LPS-dependent nos2 expression. TSA specifically inhibits LPS-dependent genes of secondary response, which require new protein synthesis for their induction but not those belonging to the primary response, which do not depend on this process. Deacetylase activity acts at the transcriptional level because RNA polymerase II was not bound after LPS stimulus when we added TSA. A link between the global acetylation caused by HDAC inhibitor and gene promoter recruitment of CDK8 was found. This Mediator complex subunit associates with Med 12, Med13, and cyclin C to form a submodule that is a transcriptional negative regulator. We also found that TSA reduces C/EBPβ phosphorylation without affecting its binding to DNA. Taken together, these results shed light on the molecular mechanisms involved in the transcriptional regulation of LPS-treated macrophages and on how TSA targets critical LPS-induced genes, such as nos2 and tnf-α, in inflammatory macrophage response.
... A direct effect of changes in erythrocyte function on the heart seems plausible, as erythrocytes both carry oxygen to tissues and have an important role in cardiovascular regulation through release of extracellular nucleotides and other mediators [27]. Erythrolysis is associated with increased release of free radicals [28], which are thought to be detrimental to the heart. Pathophysiological processes, which have been suggested to be associated with increased RDW, include oxidative stress and inflammation as these states may reduce red blood cell survival and lead to a more mixed population of erythrocytes in the circulation [29]. ...
Red blood cell distribution width (RDW), a measure of variation in erythrocyte volume, has been associated with several cardiovascular disorders, but the relationship with atrial fibrillation (AF) remains unclear. We investigated the association between RDW and incidence of first hospitalization due to AF in a population-based cohort.
RDW was measured in 27 124 subjects from the general population (age 45-73 years, 62% women) with no history of AF, heart failure, myocardial infarction or stroke. The association between baseline RDW and incidence of AF identified from the Swedish Hospital Discharge Register was evaluated.
During a mean follow-up of 13.6 years, 1894 subjects (53% men) were hospitalized with a diagnosis of AF. After adjustment for potential confounding factors, including cardiovascular disease risk factors, nutrient intake (iron, vitamin B12 and folate) and several haematological parameters (haemoglobin concentration, mean corpuscular volume and corpuscular haemoglobin content), the hazard ratio (HR) for incidence of AF was 1.33 [95% confidence interval (CI) 1.16-1.53) for the fourth versus first quartile of RDW (P for trend <0.001). The results were essentially unchanged when subjects with incident myocardial infarction or hospitalizations because of heart failure were censored from the analysis (HR 1.30, 95% CI 1.13-1.51; P for trend = 0.001).
RDW was associated with incidence of AF independently of several cardiovascular, nutritional and haematological factors in this study of middle-aged subjects from the general population. This article is protected by copyright. All rights reserved.
... Oxidative burst is usually associated with phagocytosis [51]. However, it is shown that respiratory proteins (hemoglobin and hemocyanin) can also produce ROS without phagocytosis [52]. In the light of the variation in stimulation ability, and apart from the finding that granulocytes are involved similarly in both phagocytosis and oxidative burst, it is still not clear whether the phagocytosis and oxidative burst are closely related in lungfish granulocytes. ...
... Many reports demonstrate multiple functions of hemocyanin in molluscans, such as monoxygenase (Suzuki et al., 2008), phenoloxidase (Decker and Jaenicke, 2004; Hristova et al., 2008), tyrosinase and catecholoxidase (Salvato et al., 1998; Campello et al., 2008). Furthermore, Bogdan (2007) discusses and proposes how respiratory Table 2 Amino acid composition of OmA. ...
A 66-kDa lectin (OmA) was purified from the serum of the Yucatan peninsula endemic octopus (Octopus maya) by a single step affinity chromatography on glutaraldehyde-fixed stroma from rat erythrocytes. OmA corresponds to 0.8% of the total circulating protein in the hemolymph; it is composed of three equal subunits of 22kDa each, and 7.4% of linked carbohydrates. The amino acids' composition indicated that agglutinin contained mainly aspartic and glutamic acids, and cysteine and methionine were identified in minor proportion. OmA agglutinates mainly rat, guinea pig, and rabbit erythrocytes, and this activity is partially inhibited by galactosamine, melobiose, galacturonic acid, mannose, and methyl alpha and beta galactosides. Hemagglutinating activity is not dependent on divalent cations, such as Ca(2+), Mg(2+), or Mn(2+). The OmA subunits showed no identity for any lectin in databases but partial identity with the type A hemocyanin from Octopus dolfleini hemolymph; the main similarities are related to tyrosinase domains and copper A and B sites that conform to the oxygen-binding site of hemocyanin.
... If this fails, natural killer cells and macrophages act to limit the extent of parasitaemia, by production of gammainterferon (IFN-g), tumour necrosis factor-alpha (TNF-a), nitrous oxide (NO) and reactive oxygen species (Homer et al. 2000). There is also suggested involvement of the recently discovered MetHb-pseudoperoxidase pathway (Bogdan 2007; Jiang et al. 2007). Ultimately, a resolution stage begins, with parasitaemia levels peaking and then rapidly declining due to the action of CD4CT cells and IFN-g (Igarashi et al. 1999; Homer et al. 2000). ...
Maternal experience before and during pregnancy is known to play a key role in offspring development. However, the influence of social cues about disease in the maternal environment has not been explored. We indirectly exposed pregnant mice to infected neighbours by housing them next to non-contagious conspecifics infected with Babesia microti. We examined the effect of this indirect immunological exposure on both the females and their adult offspring. Exposed females had higher levels of serum corticosterone and increased kidney growth compared with those with uninfected neighbours. These exposed females subsequently produced offspring that as adults showed an accelerated immune response to B. microti and less aggression in social groups. We suggest that ambient information regarding disease is used adaptively to maximize offspring survival and reproductive success in a challenging environment. Our results shed light on the impact of social information and maternal effects on life histories, and have important consequences for our understanding of epidemiology and individual disease susceptibility in humans and other animals. They also lead us to question the suitability of some laboratory housing conditions during experimental procedures, which may impact negatively upon both animal welfare and the validity of animal science.
This study presents a new approach for identifying myeloperoxidase (MPO) inhibitors with strong in vivo efficacy. By combining inhibitor-like rules and structure-based virtual screening, the pipeline achieved a 70% success rate in discovering diverse, nanomolar-potency reversible inhibitors and hypochlorous acid (HOCl) scavengers. Mechanistic analysis identified RL6 as a genuine MPO inhibitor and RL7 as a potent HOCl scavenger. Both compounds effectively suppressed HOCl production in cells and neutrophils, with RL6 showing a superior inhibition of neutrophil extracellular trap release (NETosis). In a gout arthritis mouse model, intraperitoneal RL6 administration reduced edema, peroxidase activity, and IL-1β levels. RL6 also exhibited oral bioavailability, significantly reducing paw edema when administered orally. This study highlights the efficacy of integrating diverse screening methods to enhance virtual screening success, validating the anti-inflammatory potential of potent inhibitors, and advancing the MPO inhibitor research.
Erythrocytes capture pathogens in circulation and present them to antigen-presenting cells (APCs) in the spleen. Senescent or apoptotic erythrocytes are physiologically eliminated by splenic APCs in a non-inflammatory manner as to not induce an immune reaction, while damaged erythrocytes tend to induce immune activation. The distinct characteristics of erythrocytes in their lifespan or different states inspire the design of targeting splenic APCs for vaccine delivery. Specifically, normal or damaged erythrocyte-driven immune targeting can induce antigen-specific immune activation, whereas senescent or apoptotic erythrocytes can be tailored to achieve antigen-specific immune tolerance. Recent studies have revealed the potential of erythrocyte-based vaccine delivery; however, there is still no in-depth review to describe the latest progress. This review summarizes the characteristics, different immune functions, and diverse vaccine delivery behaviors and biomedical applications of erythrocytes in different states. This review aims to contribute to the rational design and development of erythrocyte-based vaccine delivery systems for treating various infections, tumors, inflammatory diseases, and autoimmune diseases.
The implications of oxygen for medicine are basically of two kinds. First, there is the continued need by human tissues for an adequate supply of dioxygen; if that is not met, a condition called hypoxia may arise, with serious medical consequences. There are a wide number of causes for hypoxia, and a variety of medical responses.
An organism’s physiological equilibrium is critically reliant on its immune system, which provides protection against parasites, pathogens, toxic substances, and cancerous cells and allows recovery from injuries. The immune response is, however, not cost free. It demands various kinds of resources, but also more subtle costs. In the last couple of decades, there has been growing interest by ecophysiologists and evolutionary ecologists in the investigation of oxidative stress as a potential cost of immune activation and induction of anti- and pro-inflammatory mechanisms. A core idea of the immuno-oxidative ecology is that oxidative stress may provide a currency to quantify physiological costs that impinge on growth, reproduction or senescence. This chapter deals with such research and also examines how exposure to mild stress may stimulate activity of immune cells through hormetic mechanisms.
Sometime before 2.7 BYA, a new and biologically toxic substance began to appear in the environment. Biologically produced dioxygen, O2, probably first began to accumulate in small pools or layers above cyanobacterial mats. These photosynthesizers must have already developed ways to at least partially deal with dioxygen and, with greater difficulty, the reactive oxygen species (ROS) derived from it (see Chap. 1 and below). But for primitive anaerobes in the vicinity, these new substances must have been especially toxic. Nevertheless, it is clear that they evolved ways to cope with the new threats. One way was to simply avoid dioxygen altogether.
Examination of the cerebrospinal fluid aids in monitoring neurosurgical and neurointensive care patients, which is necessary for guiding important therapeutic interventions. This should be generally available, should be available in short time and should provide reliable information about changes in the central nervous system of these patients. The CSF compartment in neurosurgical and neurointensive care patients is very intricate. It is usually influenced by the primary damage to the central nervous system and reparative processes, which need to be differentiated from the possible infectious or non-infectious complications. A solution to this complicated situation is repeated evaluation of the CSF, with emphasis on its inflammatory nature. A cytological examination and monitoring the turnover of energy in the CSF compartment play a crucial role in this diagnostic process. Combined evaluation of these indicators based on general immunological principles enables differentiation between reparation, or the so called "clean-up reaction", from any infectious or non-infectious complications in the CNS. In this paper, we specifically focus on the nature, the causes and the detection of purulent inflammation and the detection of very intensive inflammatory process associated with oxidative burst of macrophages in the CSF. We also mention the possibilities of basic liquorology in the detection of the CSF circulation disorders, the CNS tissue destruction and bleeding into the CSF pathways.
Rhogocytes, terminal cells of protonephridia, and podocytes of metanephridial systems share an architectural feature that creates an apparent sieving device. The sieve serves to ultrafilter body fluid during the excretion and osmoregulation process carried out by nephridial systems, but its function in rhogocytes is unclear. Rhogocytes are molluscan hemocoelic cells that appear to have various functions related to metabolism of metal ions, including synthesis of hemocyanin in some gastropods and metal detoxification in pteriomorph bivalves. A hypothesis that proposed developmental and possibly evolutionary conversion between protonephridial terminal cells and rhogocytes has never been further explored; indeed, information on the occurrence of rhogocytes in molluscan developmental stages is meager. We used transmission electron microscopy to show that rhogocytes are present within larvae of eight species of gastropods sampled from the three major gastropod clades with a feeding larval stage in the life history. In larvae of a heterobranch gastropod, a rhogocyte was located next to each terminal cell of a pair of protonephridia that flanked the foregut, whereas all six species of caenogastropod larvae and a neritimorph larva that we examined had rhogocytes, but no protonephridia, in this location. We did not find ring-shaped profiles of hemocyanin decamers within rhogocytes of larvae or pre-hatch embryos. Rhogocytes in newly released larvae of Nerita melanotragus contained orderly bundles of cylinders, but the diameter of the cylinders was only 70% of the diameter typical of hemocyanin multidecamers. By examining embryos of the caenogastropod Nassarius mendicus at four successive developmental time points that bracketed the occurrence of larval hatching, we found that terminal cells from non-functional protonephridia in pre-hatch embryos transformed into rhogocytes around the time of hatching. This empirical evidence of ontogenetic transformation of protonephridial terminal cells into rhogocytes might be interpreted as developmental recapitulation of an evolutionary transition that occurred early in molluscan history.
Significance:
Inflammation and immunity can be associated with varying degrees of heme release from hemoproteins, eventually leading to cellular and tissue iron (Fe) overload, oxidative stress, and tissue damage. Presumably, these deleterious effects contribute to the pathogenesis of systemic infections.
Recent advances:
Heme release from hemoglobin sensitizes parenchyma cells to undergo programmed cell death in response to proinflammatory cytokines, such as tumor necrosis factor. This cytotoxic effect is driven by a mechanism involving intracellular accumulation of free radicals, which sustain the activation of the c-Jun N-terminal kinase (JNK) signaling transduction pathway. While heme catabolism by heme oxygenase-1 (HO-1) prevents programmed cell death, this cytoprotective effect requires the co-expression of ferritin H (heart/heavy) chain (FTH), which controls the pro-oxidant effect of labile Fe released from the protoporphyrin IX ring of heme. This antioxidant effect of FTH restrains JNK activation, whereas JNK activation inhibits FTH expression, a cross talk that controls metabolic adaptation to cellular Fe overload associated with systemic infections.
Critical issues and future directions:
Identification and characterization of the mechanisms via which FTH provides metabolic adaptation to tissue Fe overload should provide valuable information to our current understanding of the pathogenesis of systemic infections as well as other immune-mediated inflammatory diseases.
Natural selection should favour parents that are able to adjust their offspring's life-history strategy and resource allocation in response to changing environmental and social conditions. Pathogens impose particularly strong and variable selective pressure on host life histories, and parental genes will benefit if offspring are appropriately primed to meet the immunological challenges ahead. Here, we investigated transgenerational immune priming by examining reproductive resource allocation by female mice in response to direct infection with Babesia microti prior to pregnancy. Female mice previously infected with B. microti gained more weight over pregnancy, and spent more time nursing their offspring. These offspring generated an accelerated response to B. microti as adults, clearing the infection sooner and losing less weight as a result of infection. They also showed an altered hormonal response to novel social environments, decreasing instead of increasing testosterone production upon social housing. These results suggest that a dominance-resistance trade-off can be mediated by cues from the previous generation. We suggest that strategic maternal investment in response to an infection leads to increased disease resistance in the following generation. Offspring from previously infected mothers downregulate investment in acquisition of social dominance, which in natural systems would reduce access to mating opportunities. In doing so, however, they avoid the reduced disease resistance associated with increased testosterone and dominance. The benefits of accelerated clearance of infection and reduced weight loss during infection may outweigh costs associated with reduced social dominance in an environment where the risk of disease is high.
The critical review describes the known dicopper systems mediating the aromatic hydroxylation of monophenolic substrates. Such systems are of interest as structural and functional models of the type 3 copper enzyme tyrosinase, which catalyzes the ortho-hydroxylation of tyrosine to DOPA and the subsequent two-electron oxidation to dopaquinone. Small-molecule systems involving μ-η²:η² peroxo, bis-μ-oxo and trans-μ-1,2 peroxo dicopper cores are considered separately. These tyrosinase models are contrasted to copper-dioxygen systems inducing radical reactions, and the different mechanistic pathways are discussed. In addition to considering the stoichiometric conversion of phenolic substrates, the available catalytic systems are described. The second part of the review deals with tyrosinase. After an introduction on the occurrence and function of tyrosinases, several aspects of the chemical reactivity of this class of enzymes are described. The analogies between the small-molecule and the enzymatic system are considered, and the implications for the reaction pathway of tyrosinase are discussed (140 references).
Quinoid precursors of melanin and/or reactive oxygen species (ROS) generated during melanogenesis have been implicated as cytotoxic molecules in the immune responses of insects against their internal metazoan parasites. No study has yet identified the killing components produced in conjunction with melanotic encapsulation responses, or explained how cytotoxic molecules generated in the open circulatory system of an insect can selectively destroy foreign tissues. Strains of Drosophila melanogaster with differing immune capabilities against the wasp parasitoid Leptopilina boulardi were examined for superoxide anion (O2-.) formation during parasitization. Elevated levels of O2-. were produced by immune reactive (R-strain) hosts during melanotic encapsulation of the parasitoid, but not by susceptible (S-strain) hosts in which the parasitoid developed unmolested. Both a superoxide dismutase (SOD)-deficient strain (cSODn108, red/TM3/Sb Ser) and a catalase (CAT)-deficient strain (Catn1) also produced melanotic capsules and elevated levels of O2-. when infected, but these reactions were unsuccessful and the parasitoids survived, indicating that neither the quinoid precursors of melanin nor O2-. per se were cytotoxic. Immune incompetence in SOD-deficient and CAT-deficient hosts is attributed in part to defects in hydrogen peroxide (H2O2) metabolism, and/or the inability of these metalloenzyme-deficient strains to initiate the metal-mediated reductive cleavage of H2O2 required for the production of the cytotoxic hydroxyl radical (.OH). The role proposed for O2-. in Drosophila cellular immunity is one of potentiating the formation of .OH. Melanin, which contains both oxidizing and reducing components, may serve a dual role in producing O2-. and sequestering redox-active metal ions, thereby confining the production of ROS. Host-parasite susceptibility in the Drosophila-Leptopilina system may be determined by the ability of the parasitoid to modulate hemocyte activity and prevent both effective melanotic encapsulation and the generation of cytotoxic levels of ROS.
Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish β-1,3-glucan-binding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units.
The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via ionic interaction with its C-terminal region.
This interaction with a peroxidase seems to be a novel function for an SOD. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances.
Recent research has shown that myoglobin and hemoglobin play important roles in the pathology of certain disease states, such as renal dysfunction following rhabdomyolysis and vasospasm following subarachnoid hemorrhages. These pathologies are linked to the interaction of peroxides with heme proteins to initiate oxidative reactions, including generation of powerful vasoactive molecules (the isoprostanes) from free and membrane- bound lipids. This review focuses on the peroxide-induced formation of radicals, their assignment to specific protein residues, and the pseudoperoxidase and prooxidant activities of the heme proteins. The discovery of heme to protein cross-linked forms of myoglobin and hemoglobin in vivo, definitive markers of the participation of these heme proteins in oxidative reactions, and the recent results from heme oxygenase knockout/knockin animal model studies, indicate that higher oxidation states (ferryl) of heme proteins and their associated radicals play a major role in the mechanisms of pathology.
Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA) activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens.
Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.
• innate immunity
• pattern
• prophenoloxidase
• Toll
In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157-2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.
A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO-activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as beta-1,3-glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti-microbial, cytotoxic, opsonic, or encapsulation-promoting activities.
The hemocytes from the giant freshwater prawn Macrobrachium rosenbergii were examined for their ability to generate superoxide anion (O(2) (-)) in vitro upon exposure to various components derived from microbial cell wall components. Among the test molecules, laminarin (a polymer of beta-1, 3 glucans), mannan and LPS from five different bacterial species produced a differential response in terms of stimulated O(2) (-) production in prawn hemocytes, suggesting the ability of the hemocytes to differentiate non-self. This response was almost completely inhibited by superoxide dismutase (SOD) suggesting SOD-inhibitable O(2) (-) generation by prawn hemocytes. Phorbol 12-myristate 13-acetate (PMA) and Ca ionophore led to enhanced O(2) (-) generation by the hemocytes and this suggests the possible role of protein kinase C and Ca(2+) ions in such generation. Cytochemical analysis using nitroblue tetrazolium (NBT)-staining revealed the importance of granular hemocytes in O(2) (-) generation in these prawns. Inhibition of O(2) (-) generation by inhibitors of NADPH-oxidase and phenoloxidase pathways clearly reveal the involvement of two different pathways in non-self stimulated O(2) (-) generation by the prawn hemocytes. These findings demonstrate the importance of O(2) (-) generation and role of possible pathways in hemocyte mediated cellular immune response of a crustacean.
This review presents the common features and differences of the type 3 copper proteins with respect to their structure and function. In spite of these differences a common mechanism of activation and catalysis seems to have been preserved throughout evolution. In all cases the inactive proenzymes such as tyrosinase and catecholoxidase are activated by removal of an amino acid blocking the entrance channel to the active site. No other modification at the active site seems to be necessary to enable catalytic activity. Hemocyanins, the oxygen carriers in many invertebrates, also behave as silent inactive enzymes and can be activated in the same way. The molecular basis of the catalytic process is presented based on recent crystal structures of tyrosinase and hemocyanin. Minor conformational differences at the active site seem to decide about whether the active site is only able to oxidize diphenols as in catecholoxidase or if it is also able to o-hydroxylate monophenols as in tyrosinase.
Although quinone production and melanin formation are widely recognized as an integral part of the insect defense system, experimental evidence is lacking that the proteolytic activation of prophenoloxidase participates in the direct killing of invading microbes-active phenoloxidase generates quinones that polymerize to form melanin. Here, we report the antimicrobial effect of reactive intermediates produced in phenoloxidase-catalyzed reactions. After being treated with Manduca sexta phenoloxidase and dopamine, Escherichia coli and Bacillus subtilis ceased to grow, whereas the growth of Pichia pastoris was slightly affected. Microscopic analysis showed melanin deposition on cell surface, aggregation of bacteria, and loss of cell mobility. Viability tests revealed major decreases in the bacterial colony counts and, since the decrease remained significant after dispersion of the cell clumps, the reactive compounds were surmised to have aggregated and killed E. coli and B. subtilis cells. Under the experimental conditions, 60-94% of the Gram-negative bacteria (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium) and 52-99% of the Gram-positive bacteria (Bacillus cereus, B. subtilis, Micrococcus luteus, and Staphylococcus aureus) were killed. In the presence of phenoloxidase, dopamine or 5,6-dihydroxyindole (DHI) exhibited much higher antibacterial activity than L-dopa, N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) did, suggesting that DHI and its oxidation products were cytotoxic. The antifungal activity of DHI was detected using P. pastoris, Saccharomyces cerevisiae, Candida albicans, and Beauveria bassiana. These results established that prophenoloxidase activation is an integral component of the insect defense system involving a multitude of enzymes (e.g. proteinases, oxidases, and dopachrome conversion enzyme (DCE)), which immobilizes and kills invading microorganisms.
The evolution of the host-pathogen relationship comprises a series of invasive-defensive tactics elicited by both participants. The stereotype is that the antimicrobial immune response requires multistep processes. Little is known about the primordial immunosurveillance system, which probably has components that directly link sensors and effectors. Here we found that the respiratory proteins of both the horseshoe crab and human were directly activated by microbial proteases and were enhanced by pathogen-associated molecular patterns, resulting in the production of more reactive oxygen species. Hemolytic virulent pathogens, which produce proteases as invasive factors, are more susceptible to this killing mechanism. This 'shortcut' antimicrobial strategy represents a fundamental and universal mode of immunosurveillance, which has been in existence since before the split of protostomes and deuterostomes and still persists today.
Inflammation: Basic Principles and Clinical Correlates
- S J Klebanoff
- SJ Klebanoff
- M W Johannson
- MW Johannson