Mechanism by which immune complexes are deposited in hosts tissue.

We offer an explanation how immune complexes are deposited in tissues of auto-immune disorders in humans. These disorders are characterized by the accumulation in tissues of large numbers of neutrophils, which can shed out long extracellular traps (NETs) rich in a nucleosome and in highly opsonic poly cations, histone, LL37, defensins and elastase possessing properties similar to antibodies. These can bind by strong electrostatic forces to negatively charged domains in immune globulins, thus facilitating their deposition and internalization by tissue cells. However, the main cause for tissue damage in auto-immune patients is inflicted by the plethora of toxic pro-inflammatory agents released by activated neutrophils. To ameliorate tissue damage and the cytokine storms, it is recommended to administer to patients highly anionic heparins accompanied by steroids, methotrexate, colchicine, copaxone, and also by additional agents which retarded neutrophil functions.

A Novel Hypothetical Approach to Explain the Mechanisms of Pathogenicity of Rheumatic Arthritis.

The autoimmune disorder rheumatoid arthritis (RA) is a relapsing and chronic inflammatory disease that affects the synovial cells, cartilage, bone, and muscle. It is characterised by the accumulation of huge numbers of polymorphonuclear neutrophils (PMNs) and macrophages in the synovia. Auto-antibodies are deposited in the joint via the activity of highly cationic histones released from neutrophil extracellular traps (NETs) in a phenomenon termed NETosis. The cationic histones function as opsonic agents that bind to negatively charged domains in autoantibodies and complement compounds via strong electrostatic forces, facilitating their deposition and endocytosis by synovial cells. However, eventually the main cause of tissue damage is the plethora of toxic pro-inflammatory substances released by activated neutrophils recruited by cytokines. Tissue damage in RA can also be accompanied by infections which, upon bacteriolysis, release cell-wall components that are toxic to tissues. Some amelioration of the damaged cells and tissues in RA may be achieved by the use of highly anionic heparins, which can neutralize cationic histone activity, provided that these polyanions are co-administrated with anti-inflammatory drugs such as steroids, colchicine, or methotrexate, low molecular weight antioxidants, proteinase inhibitors, and phospholipase A2 inhibitors.

A novel aspect may explain the mechanisms of pathogenicity of rheumatic fever, a multifactorial, autoimmune, infectious and inflammatory disorder which "licks the joints and bites the heart": A working hypothesis.

A novel hypothesis is presented to explain the pathogenesis of the multifactorial autoimmune disorder rheumatic fever (RF). It involves a synergistic interaction among streptococcal toxins, their cell wall components, M protein, immune complexes, complement components, cationic histones. These agents can act with cationic histones released by neutrophils during NETosis and bacteriolysis and can function as opsonic agents possessing properties similar to antibodies. Cationic histones can interact by strong electrostatic forces with negatively- charged domains on immune complexes and complement components. This allows their deposition and endocytosis in the myocardium, the heart valves, and in the joints. However, the main cause of cell and tissue damage observed in RF is due to a synergism among the plethora of pro-inflammatory substances released by activated neutrophils and macrophages. Cell damage may be mitigated to some extent by anionic heparins, heparinoids, and by anti-inflammatory drugs such as corticosteroids which counteract neutrophils and macrophage chemotaxis induced by cytokines.

Longitudinal patterns of cytokine expression at the individual level in humans after laparoscopic sleeve gastrectomy.

The study of the human response to injury has been hampered by the inherent heterogeneity in the models and methods used. By studying a standard injury longitudinally, using individual patient-level analysis, we endeavoured to better describe its dynamics. We analysed clinical variables, clinical laboratory and plasma cytokines from 20 patients at five time points. Clustering analysis showed two prototype patterns of cytokine behaviour: a concordant type, where cytokines behave the same way for all patients (notably IL-0 and TNFα), and a variable type, where different patterns of expression are seen for different patients (notably IL-8, IL-6 and IL-1RA). Analysis of the cytokines at the individual patient-level showed a strong four-way correlation between IL-1RA, GCSF, MIP-1ß and MCP-1. As it holds for most patients and not just on average, this suggests that they form a network which may play a central role in the response to gastro-intestinal injuries in humans. In conclusion, the longitudinal analysis of cytokines in a standard model allowed the identification of their underlying patterns of expression. We propose that the two prototype patterns shown may reflect the mechanism that separates the common and individual aspects of the injury response.

Comparative Evaluation of Combinatory Interaction between Endocannabinoid System Compounds and Poly-L-lysine against Streptococcus mutans Growth and Biofilm Formation.

Endocannabinoid/endocannabinoid-like (EC/EC-like) are natural endogenous compounds which have been found to affect MRSA pathogenicity. Our previous studies showed that EC/EC-like was able to impair staphylococcal biofilm formation and maintenance as well as to alter biofilm-associated virulence factors. In the present study, we investigated the combinatory effect of the selected EC/EC-like with a natural antimicrobial agent, poly-L-lysine, on cariogenic bacteria Streptococcus mutans growth and biofilm formation. Among four tested EC/EC-like, only two, anandamide (AEA) and oleoylethanolamide (OEA), exhibited synergistic combinatory effect with poly-L-lysine against S. mutans. We attribute this distinct effect to differences in the fatty acid chain structure of the selected EC/EC-like compounds. Moreover, AEA exerted a specific antibiofilm mode of action against S. mutans by effecting total inhibition of biofilm formation while still allowing bacteria viability. Finally, we postulate that the presence of EC/EC-like and poly-L-lysine could enhance the permeability and efficacy of each other via hydrophobic and electrostatic interactions with the S. mutans membrane. In conclusion, we assume that a combination of endogenous natural compounds such as EC/EC-like and poly-L-lysine may benefit oral hygiene by preventing dental plaque.

Polyphenols Inhibit Candida albicans and Streptococcus mutans Biofilm Formation.

Background: Streptococcus mutans (S. mutans) and Candida albicans (C. albicans) are two major contributors to dental caries. They have a symbiotic relationship, allowing them to create an enhanced biofilm. Our goal was to examine whether two natural polyphenols (Padma hepaten (PH) and a polyphenol extraction from green tea (PPFGT)) could inhibit the caries-inducing properties of S. mutans and C. albicans. Methods: Co-species biofilms of S. mutans and C. albicans were grown in the presence of PH and PPFGT. Biofilm formation was tested spectrophotometrically. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy. Biofilm development was also tested on orthodontic surfaces (Essix) to assess biofilm inhibition ability on such an orthodontic appliance. Results: PPFGT and PH dose-dependently inhibited biofilm formation without affecting the planktonic growth. We found a significant reduction in biofilm total biomass using 0.625 mg/mL PPFGT and 0.16 mg/mL PH. A concentration of 0.31 mg/mL PPFGT and 0.16 mg/mL PH inhibited the total cell growth by 54% and EPS secretion by 81%. A reduction in biofilm formation and EPS secretion was also observed on orthodontic PVC surfaces. Conclusions: The polyphenolic extractions PPFGT and PH have an inhibitory effect on S. mutans and C. albicans biofilm formation and EPS secretion.

Pro-inflammatory agents released by pathogens, dying host cells, and neutrophils act synergistically to destroy host tissues: a working hypothesis.

We postulate that the extensive cell and tissue damage inflicted by many infectious, inflammatory and post-inflammatory episodes is an enled result of a synergism among the invading microbial agents, host neutrophils and dead and dying cells in the nidus. Microbial toxins and other metabolites along with the plethora of pro-inflammatory agents released from activated neutrophils massively recruited to the infectious sites and high levels of cationic histones, other cationic peptides, proteinases and Th1 cytokines released from activated polymorphonuclear neutrophils (PMNs) and from necrotized tissues may act in concert (synergism) to bring about cell killing and tissue destruction. Multiple, diverse interactions among the many potential pro-inflammatory moieties have been described in these complex lesions. Such infections are often seen in the skin and aerodigestive tract where the tissue is exposed to the environment, but can occur in any tissue. Commonly, the tissue-destructive infections are caused by group A streptococci, pneumococci, Staphylococcus aureus, meningococci, Escherichia coli and Shigella, although many other microbial species are seen on occasion. All these microbial agents are characterized by their ability to recruit large numbers of PMNs. Given the complex nature of the disease process, it is proposed that, to treat these multifactorial disorders, a "cocktail" of anti-inflammatory agents combined with non-bacteriolytic antibiotics and measures to counteract the critical toxic role of cationic moieties might prove more effective than a strategy based on attacking the bacteria alone.

Green Tea Polyphenols and Padma Hepaten Inhibit Candida albicans Biofilm Formation.

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogenic fungus and can cause mucosal membrane infections and invade the blood. In the oral cavity, it can ferment dietary sugars, produce organic acids and therefore has a role in caries development. In this study, we examined whether the polyphenol rich extractions Polyphenon from green tea (PPFGT) and Padma Hepaten (PH) can inhibit the caries-inducing properties of C. albicans. Biofilms of C. albicans were grown in the presence of PPFGT and PH. Formation of biofilms was tested spectrophotometrically after crystal violet staining. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy (CSLM). Treated C. albicans morphology was demonstrated using scanning electron microscopy (SEM). Expression of virulence-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such appliances. Both PPFGT and PH dose-dependently inhibited biofilm formation, with no inhibition on planktonic growth. The strongest inhibition was obtained using the combination of the substances. Crystal violet staining showed a significant reduction of 45% in biofilm formation using a concentration of 2.5mg/ml PPFGT and 0.16mg/ml PH. A concentration of 1.25 mg/ml PPFGT and 0.16 mg/ml PH inhibited candidal growth by 88% and EPS secretion by 74% according to CSLM. A reduction in biofilm formation and in the transition from yeast to hyphal morphotype was observed using SEM. A strong reduction was found in the expression of hwp1, eap1, and als3 virulence associated genes. These results demonstrate the inhibitory effect of natural PPFGT polyphenolic extraction on C. albicans biofilm formation and EPS secretion, alone and together with PH. In an era of increased drug resistance, the use of phytomedicine to constrain biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

Effect of fixed orthodontic appliances on nonmicrobial salivary parameters.

OBJECTIVES:: To examine possible changes in the levels of salivary antioxidants, C-reactive protein (CRP), cortisol, pH, proteins, and blood in patients treated with fixed orthodontic appliances. MATERIALS AND METHODS:: Salivary samples from 21 orthodontic patients who met specific inclusion criteria were collected before the beginning of orthodontic treatment (T0; baseline), 1 hour after bonding (T1), and 4-6 weeks after bonding (T2). Oxidant-scavenging ability (OSA) was quantified using a luminol-dependent chemiluminescence assay. Cortisol and CRP levels were measured using immunoassay kits. pH levels and presence of proteins and blood in the samples were quantified using strip-based tests. RESULTS:: A significant decrease in salivary pH was observed after bonding ( P = .013). An increase in oxidant-scavenging abilities during orthodontic treatment was detected, but the change was not statistically significant. Cortisol and CRP levels slightly increased after bonding, but the difference was small without statistical significance. Changes in the presence of proteins and blood were also insignificant. CONCLUSIONS:: Exposure to fixed orthodontic appliances did not show a significant effect on salivary parameters related to inflammation or stress, with the exception of a significant but transient pH decrease after bonding.

Bacteriolysis - a mere laboratory curiosity?

The role of bacteriolysis in the pathophysiology of microbial infections dates back to 1893 when Buchner and Pfeiffer reported for the first time the lysis of bacteria by immune serum and related this phenomenon to the immune response. Later on, basic anti-microbial peptides and certain beta-lactam antibiotics have been shown not only to kill microorganisms but also to induce bacteriolysis and the release of cell-wall components. In 2009, a novel paradigm was offered suggesting that the main cause of death in sepsis is due to the exclusive release from activated human phagocytic neutrophils (PMNs) traps adhering upon endothelial cells of highly toxic nuclear histone. Since activated PMNs also release a plethora of pro-inflammatory agonists, it stands to reason that these may act in synergy with histone to damage cells. Since certain beta lactam antibiotics may induce bacteriolysis, it is questioned whether these may aggravate sepsis patient's condition. Enigmatically, since the term bacteriolysis and its possible involvement in sepsis is hardly ever mentioned in the extensive clinical articles and reviews dealing with critical care, we hereby aim to refresh the concept of bacteriolysis and its possible role in the pathogenesis of post infectious sequelae.

Combined antioxidant effects of Neem extract, bacteria, red blood cells and Lysozyme: possible relation to periodontal disease.

BACKGROUND: The common usage of chewing sticks prepared from Neem tree (Azadirachta indica) in India suggests its potential efficacy in periodontal diseases. The objective of this study is to explore the antibacterial effects of Neem leaf extract on the periodontophatic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and its antioxidant capacities alone and in combination with bacteria and polycationic peptides that may be at the site of inflammation. METHODS: Neem leaf extract was prepared by ethanol extraction. The growth kinetics of P. gingivalis and F. nucleatum under anaerobic conditions in the presence of Neem leaf extract were measured. Broth microdilution test was used to determine the Minimal Inhibitory Concentration (MIC) of Neem leaf extract against each bacterial strain. The effect of Neem leaf extract on the coaggregation of the bacteria was assessed by a visual semi-quantitative assay. The antioxidant capacities of Neem leaf extract alone and in combination with bacteria, with the addition of red blood cells or the polycationic peptides chlorhexidine and lisozyme, were determined using a chemiluminescence assay. RESULTS: Neem leaf extract showed prominent dose-dependent antibacterial activity against P. gingivalis, however, had no effect on the growth of F. nucleatum nor on the coaggregation of the two bacteria. Yet, it showed intense antioxidant activity, which was amplified following adherence to bacteria and with the addition of red blood cells or the polycationic peptides. CONCLUSIONS: Neem leaf extract, containing polyphenols that adhere to oral surfaces, have the potential to provide long-lasting antibacterial as well as synergic antioxidant activities when in complex with bacteria, red blood cells and lisozyme. Thus, it might be especially effective in periodontal diseases.

Serum histones as biomarkers of the severity of heatstroke in dogs.

Heatstroke is associated with systemic inflammatory response syndrome, leading to multiple organ dysfunction and death. Currently, there is no specific treatment decreasing hyperthermia-induced inflammatory/hemostatic derangements. Emerging studies indicate that histones leaking from damaged cells into the extracellular space are toxic, pro-inflammatory, and pro-thrombotic. We therefore hypothesize that serum histones (sHs) are elevated during heatstroke and are associated with the severity of the disease. Sixteen dogs with heatstroke and seven healthy controls were included in the study. Median serum histones (sHs) upon admission in dogs with heatstroke were significantly higher (P = 0.043) compared to that in seven controls (13.2 vs. 7.3 ng/mL, respectively). sHs level was significantly higher among non-survivors and among dogs with severe hemostatic derangement (P = 0.049, median 21.4 ng/mL vs. median 8.16 ng/mL and P = 0.038, 19.0 vs. 7.0 ng/mL, respectively). There were significant positive correlation between sHs and urea (r = 0.8, P = 0.02); total CO2 (r = 0.661, P = 0.05); CK (r = 0.678, P = 0.04); and prothrombin time (PT) 12 h post presentation (r = 0.888, P = 0.04). The significant positive correlation between sHs and other heatstroke severity biomarkers, and significant increase among severely affected dogs, implies its role in inflammation/oxidation/coagulation during heatstroke. sHs, unlike other prognostic and severity biomarkers in heatstroke, can be pharmacologically manipulated, offering a potential therapeutic target.

From amino acids polymers, antimicrobial peptides, and histones, to their possible role in the pathogenesis of septic shock: a historical perspective.

This paper describes the evolution of our understanding of the biological role played by synthetic and natural antimicrobial cationic peptides and by the highly basic nuclear histones as modulators of infection, postinfectious sequelae, trauma, and coagulation phenomena. The authors discuss the effects of the synthetic polymers of basic poly α amino acids, poly l-lysine, and poly l-arginine on blood coagulation, fibrinolysis, bacterial killing, and blood vessels; the properties of natural and synthetic antimicrobial cationic peptides as potential replacements or adjuncts to antibiotics; polycations as opsonizing agents promoting endocytosis/phagocytosis; polycations and muramidases as activators of autolytic wall enzymes in bacteria, causing bacteriolysis and tissue damage; and polycations and nuclear histones as potential virulence factors and as markers of sepsis, septic shock, disseminated intravasclar coagulopathy, acute lung injury, pancreatitis, trauma, and other additional clinical disorders.

Nuclear histones: major virulence factors or just additional early sepsis markers? A comment.

In 2009, Xu et al. and Chaput et al. in Nature Medicine had argued that the main cause of death in sepsis is the release from neutrophil nets of nuclear histone, highly toxic to endothelial cells and that these polycations are major and unique virulence factors. Since 2009, numerous researchers have also suggested the involvement of histones in the pathophysiology of many clinical disorders. If histones are indeed major unique virulence toxic agents, then heparin, activated protein C and antibodies to histone should prove excellent antisepsis agents. However, this is provided that these agents are administered to patients early enough before the activation of the cytokine storms, immune responses and the coagulation cascades are irreversibly unleashed. This may not be practical, since a diagnosis of sepsis is usually made much later. Future identifications of novel early markers are therefore needed and a compilation of cocktails of antagonists may replace the faulty single antagonists tried for many years, but in vain, to prevent death in sepsis.

Thiazolidinedione-8 Alters Symbiotic Relationship in C. albicans-S. mutans Dual Species Biofilm.

The small molecule, thiazolidinedione-8 (S-8) was shown to impair biofilm formation of various microbial pathogens, including the fungus Candida albicans and Streptococcus mutans. Previously, we have evaluated the specific molecular mode of S-8 action against C. albicans biofilm-associated pathogenicity. In this study we investigated the influence of S-8 on dual species, C. albicans-S. mutans biofilm. We show that in the presence of S-8 a reduction of the co-species biofilm formation occurred with a major effect on C. albicans. Biofilm biomass and exopolysaccharide (EPS) production were significantly reduced by S-8. Moreover, the agent caused oxidative stress associated with a strong induction of reactive oxygen species and hydrogen peroxide uptake inhibition by a mixed biofilm. In addition, S-8 altered symbiotic relationship between these species by a complex mechanism. Streptococcal genes associated with quorum sensing (QS) (comDE and luxS), EPS production (gtfBCD and gbpB), as well as genes related to protection against oxidative stress (nox and sodA) were markedly upregulated by S-8. In contrast, fungal genes related to hyphae formation (hwp1), adhesion (als3), hydrophobicity (csh1), and oxidative stress response (sod1, sod2, and cat1) were downregulated in the presence of S-8. In addition, ywp1 gene associated with yeast form of C. albicans was induced by S-8, which is correlated with appearance of mostly yeast cells in S-8 treated dual species biofilms. We concluded that S-8 disturbs symbiotic balance between C. albicans and S. mutans in dual species biofilm.

Intravesical administration of green tea extract attenuates the inflammatory response of bacterial cystitis--a rat model.

OBJECTIVE: To explore the effect of intravesical instillation of green tea extract (GTE) on a rat model of bacterial cystitis. MATERIALS AND METHODS: In vitro bactericidal properties of GTE were analysed by adding GTE to a suspension of uropathogenic E. coli (UPEC), streaking on MacConkey agar, and incubating overnight. In vivo effects of intravesical instillation of GTE on bacterial cystitis was analysed using a rat model of bacterial cystitis. In all, 42 female Sabra rats weighing 200-260 g were divided into five groups. Parameters measured were bladder weight (percentage of the total rat weight), dipstick urine analysis and histopathological changes in the bladder. Histological changes evaluated were degree of oedema, mixed inflammatory infiltration, urothelial epithelial invasion by neutrophils and reactive atypia. RESULTS: No in vitro bactericidal activity was detected for GTE. Intravesical instillation of GTE did not cause damage to the rat bladders. Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis in this rat model. CONCLUSIONS: Intravesical instillation of GTE attenuated the inflammatory response to UPEC-SR71-induced bacterial cystitis and is a novel approach to the treatment of bacterial cystitis. High concentrations of intravesical GTE did not cause histologically evident damage to the rat bladder. The results of this study are preliminary and further studies will be needed to explore the feasibility of using this approach in humans.

LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

The oxidant-scavenging abilities in the oral cavity may be regulated by a collaboration among antioxidants in saliva, microorganisms, blood cells and polyphenols: a chemiluminescence-based study.

Saliva has become a central research issue in oral physiology and pathology. Over the evolution, the oral cavity has evolved the antioxidants uric acid, ascorbate reduced glutathione, plasma-derived albumin and antioxidants polyphenols from nutrients that are delivered to the oral cavity. However, blood cells extravasated from injured capillaries in gingival pathologies, or following tooth brushing and use of tooth picks, may attenuate the toxic activities of H2O2 generated by oral streptococci and by oxidants generated by activated phagocytes. Employing a highly sensitive luminol-dependent chemiluminescence, the DPPH radical and XTT assays to quantify oxidant-scavenging abilities (OSA), we show that saliva can strongly decompose both oxygen and nitrogen species. However, lipophilic antioxidant polyphenols in plants, which are poorly soluble in water and therefore not fully available as effective antioxidants, can nevertheless be solubilized either by small amounts of ethanol, whole saliva or also by salivary albumin and mucin. Plant-derived polyphenols can also act in collaboration with whole saliva, human red blood cells, platelets, and also with catalase-positive microorganisms to decompose reactive oxygen species (ROS). Furthermore, polyphenols from nutrient can avidly adhere to mucosal surfaces, are retained there for long periods and may function as a "slow-release devises" capable of affecting the redox status in the oral cavity. The OSA of saliva is due to the sum result of low molecular weight antioxidants, albumin, polyphenols from nutrients, blood elements and microbial antioxidants. Taken together, saliva and its antioxidants are considered regulators of the redox status in the oral cavity under physiological and pathological conditions.