The impact of TLR7 agonist R848 treatment on mast cell phenotype and activity.

Bearing in mind that mast cell contribution to viral clearance is still not fully understood, in this study, we evaluated the effect of Toll-like receptor (TLR)7 viral single-stranded ribonucleic acid (ssRNA) mimic ligand, namely resiquimod (R)848, on mast cell phenotype and activity. We demonstrated that rat peritoneal mast cells exhibit surface and intracellular expression of ssRNA-specific TLR7 molecule, and that mimic ligand switches the self-expression of this receptor. We also detected other proteins associated with the cellular antiviral response: interferon-alpha receptor 1 (IFNAR1), interferon-gamma receptor 1 (IFNGR1), and major histocompatibility complex I (MHC I). Moreover, we showed that R848 caused the decrease of all molecule's expression after prolonged incubation. Interestingly, we found that R848 induced the increase of high-affinity IgE receptor (FcεRI) expression. Finally, we documented that TLR7 ligand-stimulated mast cells synthesize/release interferon (IFN)-α and -ß, tumor necrosis factor (TNF), and chemokines CCL3, CXCL8, as well as pro-inflammatory lipid mediators. Our findings confirm that mast cells may respond to TLR7 ligand by altering their phenotype and synthesizing mediators and could serve as active participants in the antiviral immune response.

Mast cell phenotypic plasticity and their activity under the influence of cathelicidin-related antimicrobial peptide (CRAMP) and IL-33 alarmins.

Invading pathogens are contained/eliminated by orchestrated actions of different humoral components of the innate immune response. One of them is endogenous molecules called alarmins, which contribute to diverse processes from danger sense until the infection extinction. Considering the participation of mast cells (MCs) in many aspects of the body's defense and, on the other hand, the importance of alarmins as molecules that signal damage/danger, in this study, we evaluated the effect of alarmins on MC phenotype and activity. We found that cathelicidin CRAMP and cytokine IL-33 significantly affect the appearance of Dectin-1, Dectin-2, RIG-I, and NOD1 receptors in mature MCs and modulate their inflammatory response. We established that chosen alarmins might stimulate MCs to release pro-inflammatory and immunoregulatory mediators and induce a migratory response. In conclusion, our data highlight that alarmins CRAMP and IL-33 might strongly influence MC features and activity, mainly by strengthening their role in the inflammatory mechanisms and controlling the activity of cells participating in antimicrobial processes.

Analysis of IL-1ß, CXCL8, and TNF-α levels in the crevicular fluid of patients with periodontitis or healthy implants.

BACKGROUND: Our study aimed to assess the level of IL-1ß, CXCL8, and TNF-α in peri-implant sulcular fluid (PISF) collected from patients with no clinical symptoms of mucositis or peri-implantitis and compare them with cytokine concentration in gingival crevicular fluid (GCF) acquired from patients with healthy periodontium and those with varying severity of periodontitis. METHODS: A total of 189 subjects were included in the study, and GCF/PISF samples were checked for IL-1ß, CXCL8, and TNF-α levels using an ELISA test. RESULTS: The IL-1ß level in PISF in patients with implants was significantly lower than in GCF in patients with mild, moderate, or severe periodontitis. The CXCL8 level in PISF was considerably lower than in patients with moderate periodontitis. The TNF-α level in PISF in patients with implants was markedly higher compared to subjects with healthy periodontium or patients with mild periodontitis. CONCLUSION: Analysis of cytokine levels may help describe the pathogenesis and early diagnosis of peri-implantitis and prevision in high-risk patients.

Curdlan stimulates tissue mast cells to synthesize pro-inflammatory mediators, generate ROS, and migrate via Dectin-1 receptor.

Mast cells (MCs) are engaged in host defense against various pathogens as they are equipped with pattern recognition receptors (PRRs). Among PRRs expressed on MCs, there are also molecules recognizing components of the fungal cell wall, which are able to induce cellular activation and response. However, little information is available concerning the MC activation by various fungal-derived components. The aim of the study was to determine whether curdlan, a model fungal particle of ß-(1,3)-glucan, can directly stimulate tissue MCs. We demonstrated that curdlan triggers MCs to initiate pro-inflammatory response as it activates these cells to synthesize essential pro-inflammatory and/or immunoregulatory factors. We also showed that curdlan serves as a potent chemoattractant for MCs and stimulates those cells to generate reactive oxygen species (ROS). Finally, we documented that curdlan induces MC response via Dectin-1. Our observations support the idea that MCs serve as important sentinels modulating immune response during fungal infection.

Adipocytokines leptin and adiponectin function as mast cell activity modulators.

A growing body of data indicates that adipocytokines, including leptin and adiponectin, are critical components not only of metabolic regulation but also of the immune system, mainly by influencing the activity of cells participating in immunological and inflammatory processes. As mast cells (MCs) are the key players in the course of those mechanisms, this study aimed to evaluate the impact of leptin and adiponectin on some aspects of MC activity. We documented that in vivo differentiated mature tissue MCs from the rat peritoneal cavity express a receptor for leptin (OB-R), as well as receptors for adiponectin (AdipoR1 and AdipoR2). We established that leptin, but not adiponectin, stimulates MCs to release of histamine as well as to generation of cysteinyl leukotrienes (cysLTs) and chemokine CCL2. We also found that both adipocytokines affect mRNA expression of various cytokines/chemokines. Leptin and adiponectin also activate MCs to produce reactive oxygen species. Moreover, we documented that leptin significantly augments the surface expression of receptors for cysLTs, i.e. CYSLTR1, CYSLTR2, and GPR17 on MCs, while adiponectin increases only GPR17 expression, and decreases CYSLTR2. Finally, we showed that both adipocytokines serve as potent chemoattractants for MCs. In intracellular signaling in MCs activated by leptin Janus-activated kinase 2, phospholipase C, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK1/2), and p38 molecules play a part whereas the adiponectin-induced activity of MCs is mediated through PI3K, p38, and ERK1/2 pathways. Our observations that leptin and adiponectin regulate MC activity might indicate that adipocytokines modulate the different processes in which MCs are involved.

Expression of Toll-like receptors 2 and 4 on peripheral mononuclear cells (PBMCs) after laparoscopic cholecystectomy.

Increasing evidence suggests that the course and intensity of inflammation, as well as repair processes, developed in response to stress, injury, and trauma, depend on the interaction between immediately released endogenous molecules, called alarmins or danger/damage-associated molecular patterns (DAMPs) and cellular pattern recognition receptors (PRR) including Toll-like receptors (TLRs) and activation of inflammatory/immune cells. Therefore, the aim of this study was to examine the expression of TLRs in peripheral blood mononuclear cells (PBMCs), CD3+, and CD14+ cells in control group and in patients before the laparoscopic cholecystectomy, and three and seven days after surgery. Flow cytometry was used to evaluate expression of TLR2 and TLR4. TLR2 and especially TLR4 expression levels on PBMCs were significantly lower in patients with asymptomatic cholelithiasis than in the control group. Laparoscopic surgery did not induce the significant changes in the expression of TLR2, both on PBMCs and CD3+ and CD14+ cell subpopulations. On the contrary, TLR4 expression level on PBMCs was significantly lower on the third and seventh postoperative day than before surgery. Collectively, the expression levels of cellular TLRs, and especially TLR2 and TLR4, might strongly influence the responsiveness of cells to DAMP activation, and in this way can regulate the intensity of inflammatory response to surgical injury.

An overview of mast cell pattern recognition receptors.

BACKGROUND: Mast cells (MCs) are long-lived immune cells of the connective tissue which play a key role in development and amplification of inflammatory process initiated inter alia by allergic reactions or microbial infections. They reside in strategic locations in the body that are notably exposed to deleterious factors disturbing homeostasis, which enables them to become one of the first-line defense strategy. MCs have developed a wide range of various mechanisms to deal with invading intruders and harmful endogenic factors. Those include storage and synthesis with a subsequent release of inflammatory mediators, forming of MC-extracellular traps, and phagocytosis. FINDINGS: Particularly, important role in microbial sensing is achieved due to the presence of different pattern recognition receptors (PRRs). The best-described receptors are Toll-like receptors activated by different pathogen- and damage-associated molecular patterns. However, MCs express also C-type lectin receptors specialized in antifungal defense, NOD-like receptors detecting bacterial peptidoglycans, and RIG-like receptors relevant in viral sensing. CONCLUSION: This review will focus on the current knowledge of PRRs expressed within different types of MCs.

Leptin stimulates tissue rat mast cell pro-inflammatory activity and migratory response.

OBJECTIVE: The aim of this study was to determine whether leptin, a member of the adipocytokines involved in immune and inflammatory response regulation, may influence some aspects of mast cell biology. MATERIALS AND METHODS: Experiments were done in vitro on fully mature tissue rat mast cells isolated from the peritoneal cavity, and leptin was used at concentrations 0.001-100 ng/ml. The effect of leptin on mast cell degranulation (histamine release assay), intracellular Ca2+ level (fluorimetry), pro-inflammatory mediator release (ELISA technique), surface receptor expression (flow cytometry and confocal microscopy), and migration (Boyden microchamber assay) was estimated. RESULTS: Leptin was found to stimulate mast cells to degranulation and histamine release. It induced the intracellular Ca2+ increase, as well. In response to leptin stimulation, mast cells generated and released cysLTs and chemokine CCL3. Leptin-induced upregulation of CYSLTR1 and CYSLTR2 surface expression was observed. Moreover, this adipocytokine stimulated mast cells to migratory response, even in the absence of extracellular matrix (ECM) proteins. CONCLUSIONS: Our observations clearly documented that leptin promotes the pro-inflammatory activity of mast cells, and it thereby engages these cells in the inflammatory processes.

Presence of archaea and selected bacteria in infected root canal systems.

Infections of the root canal have polymicrobial etiology. The main group of microflora in the infected pulp is bacteria. There is limited data that archaea may be present in infected pulp tissue. The aim of this study was to check the prevalence of archaea in necrotic root canal samples obtained from patients with primary or post-treatment infection. The prevalence of selected bacteria species (Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Synergistes sp.) in necrotic samples was evaluated as well. Sixty-four samples from root canal were collected for DNA and RNA extraction. A PCR assay based on the 16S rRNA gene was used to determine the presence of archaea and selected bacteria. Of the 64 samples, 6 were analyzed by semiquantitative reverse transcription PCR to estimate expression profiles of 16S rRNA, and another 9 were selected for direct sequencing. Archaea were detected in 48.4% samples. Statistical analysis indicated a negative association in coexistence between archaea and Treponema denticola (P < 0.05; Pearson's χ2 test). The main representative of the Archaea domain found in infected pulp tissue was Methanobrevibacter oralis. Archaea 16S rRNA gene expression was significantly lower than Synergistes sp., Porphyromonas gingivalis, and Tannerella forsythia (P < 0.05; Student's t test). Thus, it can be hypothesized that archaea may participate in the endodontic microbial community.

Body composition does not affect serum levels of cathelicidin LL-37 in elderly women with unipolar depression.

OBJECTIVES: Antimicrobial peptides are components of the innate immune system. Cathelicidin LL-37 plays an important role in antimicrobial defense, exerts proinflammatory effect and strongly affects the immune system functioning. Our recent study revealed that serum concentration of LL-37 is increased in elderly women with depression. The aim of this study is to evaluate serum LL-37 levels in elderly women with depression and to compare them with non-depressed elderly women, matched for anthropometric and body composition parameters. METHODS: Forty women with unipolar depression and 23 non-depressed women (age ≥60 years) were included into the study. Anthropometric measurements and biochemical analyzes were performed. Concentration of LL-37 in serum was assessed using ELISA method. Body composition was measured using two methods: bioimpedance analysis (BIA) and dual-energy X-ray absorptiometry (DXA). RESULTS: There was a statistically significant difference (p =.038) in serum LL-37 level between patients with depression (3.55 ± 6.57 ng/mL) and control subjects (2.01 ± 3.88 ng/mL). Apart from visceral adipose tissue mass (%) in the depression group, we found no associations between serum LL-37 and analyzed anthropometric or body composition parameters. CONCLUSIONS: Results of this study indicate that with the exception of visceral adipose tissue, LL-37 serum levels are not affected by anthropometric or body composition parameters. The association between visceral adipose tissue and LL-37 may indicate that visceral fat could be responsible for the increased LL-37 production.

Serum concentrations of antimicrobial peptide cathelicidin LL-37 in patients with bacterial lung infections.

Nowadays, data indicate that antimicrobial peptides play an important role in immunological defense. Human cathelicidin LL-37 possesses a broad spectrum of antimicrobial properties against Gram-positive and Gram-negative bacteria, and is thereby an important component of defense mechanisms within the respiratory tract. In this study, we determined the LL-37 serum level in patients with pneumonia caused by different bacteria species in comparison with healthy subjects. Twenty-two patients with pneumonia caused by coccal Gram-positive bacteria (I), 16 patients with pneumonia caused by Haemophilus influenzae (II), 29 patients with pneumonia caused by members of the Enterobacteriaceae (III), 13 patients caused by non-fermenting Gram-negative bacteria (IV), and 30 healthy controls were enrolled in the study. Serum LL-37 concentration was measured using an enzyme-linked immunosorbent assay (ELISA). The mean LL-37 concentration in pneumonia patients was significantly higher in group I (p = 0.0032), group II (p = 0.0022), and group III (p = 0.019), and significantly lower in group IV (p = 0.000004) as compared with healthy volunteers. Our data suggest that LL-37 plays an important role in defense mechanisms during pneumonia. The reduced level of this peptide in subjects with pneumonia caused by opportunistic bacteria may reflect weakened immune system reactivity in these patients.

Leukotriene receptor expression in mast cells is affected by their agonists.

The effects of LTs are mediated by GPCRs: cysLTs interact with CYSLTR1, CYSLTR2, or GPR17, and LTB4 acts via BLT1R or BLT2R. Data relating to the presence of these receptors in mature tissue mast cells are not entirely known. By confocal microscopy with image analyses and flow cytometry, we established that native rat mast cells isolated from peritoneal cavity constitutively express all studied receptors. Moreover, we clearly documented that LTs by themselves can influence their own receptor expression. Low concentrations of LTs induce translocation of LT receptors from cell interior to plasma membrane, which can lead to increased mast cell responsiveness to LT stimulation. High concentrations of LTs cause internalization and, in consequence, reduction in the number of receptors on the cell surface, and it may result in desensitization of mast cells to subsequent LT stimulation. These observations may imply a physiological feedback mechanism regulating mast cell sensitivity to LT activation within tissues.

Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international inter-laboratory scoring exercise.

The buccal micronucleus cytome (BMNcyt) assay in uncultured exfoliated epithelial cells from oral mucosa is widely applied in biomonitoring human exposures to genotoxic agents and is also proposed as a suitable test for prescreening and follow-up of precancerous oral lesions. The main limitation of the assay is the large variability observed in the baseline values of micronuclei (MNi) and other nuclear anomalies mainly related to different scoring criteria. The aim of this international collaborative study, involving laboratories with different level of experience, was to evaluate the inter- and intra-laboratory variations in the BMNcyt parameters, using recently implemented guidelines, in scoring cells from the same pooled samples obtained from healthy subjects (control group) and from cancer patients undergoing radiotherapy (treated group). The results indicate that all laboratories correctly discriminated samples from the two groups by a significant increase of micronucleus (MN) and nuclear bud (NBUD) frequencies and differentiated binucleated (BN) cells, associated with the exposure to ionizing radiation. The experience of the laboratories was shown to play an important role in the identification of the different cell types and nuclear anomalies. MN frequency in differentiated mononucleated (MONO) and BN cells showed the greatest consistency among the laboratories and low variability was also detected in the frequencies of MONO and BN cells. A larger variability was observed in classifying the different cell types, indicating the subjectivity in the interpretation of some of the scoring criteria while reproducibility of the results between scoring sessions was very good. An inter-laboratory calibration exercise is strongly recommended before starting studies with BMNcyt assay involving multiple research centers.

Circulating cathelicidin LL-37 in adult patients with pulmonary infectious diseases.

PURPOSE: The antimicrobial peptide cathelicidin LL-37 plays a role in the immune response in the course of lung infections; however, the exact role of LL-37 in defense mechanisms against bacteria within the respiratory tract is has not been precisely described. The aim of our study was to evaluate LL-37 concentrations in the serum of pulmonary tuberculosis (TB) patients, patients with pneumonia caused by Gram-positive and Gram-negative bacteria and to compare them with those of healthy subjects. METHODS: Thirty TB patients, 30 patients with pneumonia caused by Gram-positive bacteria, 30 patients with pneumonia caused by Gram-negative bacteria, and 30 healthy control subjects were enrolled in the study. Serum LL-37 concentration was measured using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean (± SEM) LL-37 concentration in patients with TB (13.94±5.13 ng/mL) was significantly higher than that in patients with Gram-positive bacteria-induced pneumonia (7.87±4.58 ng/mL, P=0.00077), in patients with Gram-negative bacteria-induced pneumonia (10.27±3.60 ng/mL, P=0.00730), and in control healthy subjects (1.75±0.71 ng/mL, P=0.00004). CONCLUSION: Our data suggest that cathelicidin LL-37 is an important element of host defense in the course of bacterial diseases within the respiratory tract, particularly when the infection is caused by an intracellular pathogen.

Evaluation of Metalloproteinase-8 Levels in Crevicular Fluid of Patients with Healthy Implants or Periodontitis.

Evaluation of periodontal and peri-implant tissue condition is mainly based on clinical examination and imaging diagnostics. Some data imply that Metalloproteinase-8 (MMP-8) level examination in peri-implant sulcular fluid (PISF) might be useful for evaluating the condition of peri-implant tissues and monitoring a development of peri-implant inflammation, including both mucositis and peri-implantitis. Hence, in this study, we decided to evaluate the level of MMP-8 in PISF obtained from patients without clinical symptoms of mucositis or peri-implantitis and compare it with MMP-8 level in gingival crevicular fluid (GCF) obtained from patients with healthy periodontium and those with varying severity of periodontitis. A total of 189 subjects were included in the study, and GCF/PISF samples were analysed for MMP-8 level by ELISA test. We documented that MMP-8 level in PISF obtained from patients without symptoms of mucositis or peri-implantitis was significantly higher not only than in GCF of periodontally healthy patients but also, which seems to be very interesting, than in GCF of patients with varying degrees of periodontal inflammation, consistent with earlier studies. Our observation might imply that monitoring of MMP-8 level in PISF could help to diagnose mucositis/peri-implantitis in an early stage, prior to clinical manifestations, which may allow for quick start of appropriate therapy.

Mast cells as the strength of the inflammatory process.

The inflammatory process is a complex host defence mechanism aimed at the elimination of deleterious factors disturbing homeostasis. Inflammation consists of several interdependent stages controlled by a wide range of mediators. Those include acute phase proteins, heat shock proteins, complement components, biogenic amines, cytokines, lipid-derived mediators, reactive oxygen species, nitric oxide, proteolytic enzymes, and kinins. Due to the strategic location in the body, mast cells play a protective role in the inflammatory process, through its initiation, amplification, and resolution. Mast cells degranulate and/or newly produce, and release various mediators classified into three groups: preformed mediators, de novo synthesised lipid mediators, and newly synthesised cytokines. Those mediators have an impact on different processes occurring during inflammation, inter alia, they influence blood vessels leading to dilation, enhanced adhesion molecule expression, and increased permeability. Furthermore, mast cell mediators play a pivotal role in inflammatory cell chemotaxis, degradation of extracellular matrix proteins, impact on stationery cells and resolution of inflammation. The release of mast cell mediators and their actions constitute a highly complex and still not fully understood mechanism, which warrants further studies of the action of mast cells in inflammation. This review will focus on the current knowledge concerning the broad role of mast cells in the inflammatory process.

Cathelicidins and defensins regulate mast cell antimicrobial activity.

Cathelicidins and defensins are the multifunctional host defense molecules essential for immune response to infection. In recent years they have been shown to be natural, broad-spectrum antimicrobials against both Gram-positive and Gram-negative bacteria, enveloped viruses, and fungi. These small peptides kill the invaded pathogens by destroying their cell membranes and can neutralize biological activities of endotoxin. Apart from exerting direct antimicrobial effects, cathelicidins and defensins can also trigger innate and adaptive defense responses in the host. The functions of the host derived peptides in immunomodulation have been also investigated. Reported activities of these peptides include chemoattractant function, inhibition of neutrophil apoptosis, and ROS production. These peptides directly activate inflammatory cells to production and release of different pro-inflammatory and immunoregulatory mediators, cytokines, and chemokines, however cathelicidins and defensins might mediate the generation of anti-inflammatory cytokines, as well. Insights into the miscellaneous functions of mast cells have exposed that they possess the ability to respond to pathogens and modulate immune response. These immune sentinel cells play a pivotal role in defense mechanisms mainly through the presence of pattern recognition receptors and by release different preformed and newly synthesized mediators and cytokines. The present review provides an introduction to the field of cathelicidins and defensins in general and discusses their impact on mast cells.

[Alternatives for antibiotics - antimicrobial peptides and phages].

The constant increase in the number of bacteria resistant to antibiotics poses a substantial problem for the therapy of infectious diseases of different etiologies. The growing insensitivity of pathogens on the classical methods of treatment is associated mainly with multiple mechanisms of resistance created by bacteria. Furthermore, no proper antibiotic treatment causes the appearance of resistant strains even at the last line drugs. Therefore, there are still being sought alternatives in the treatment of difficult to eradicate pathogens. The antimicrobial peptides including cathelicidins, defensins, lysozyme, lactoferrin, histatins and bacteriocins arouse huge interest as potential therapeutics. They exhibit a broad spectrum of activity against many Gram-positive and Gram-negative bacteria, but also against fungi. Moreover, they are considered much safer than antibiotics, due to the fact that they are present in all eukaryotic organisms, in which they are an essential element of the immune system. In addition, phage therapy is also strongly recommended as alternative antibacterial approach. In this review we highlight the potential uses of antimicrobial peptides and bacteriophages in the treatment of infections of various etiologies.

Expression of surface and intracellular Toll-like receptors by mature mast cells.

Nowadays, more and more data indicate that mast cells play an important role in host defense against pathogens. That is why it is essential to understand the expression of Toll-like receptors (TLRs) by mast cells, because these molecules play particularly significant role in initiation host defense against microorganisms as they recognize both wide range of microbial pathogen-associated molecular patterns (PAMPs) and various endogenous damage-associated molecular patterns (DAMPs) released in response to infection. Therefore, we examined the constitutive expression of both surface and endosomal TLRs in rat native fully mature tissue mast cells. By the use of qRT-PCR we found that these cells express mRNAs for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9. The expression of TLR3, TLR4, TLR5, TLR7, and TLR9 transcripts were low and comparable and only the expression of TLR2 transcript was significant. By the use of flow cytometry technique, we clearly documented that mast cells express TLR2, TLR4, and TLR5 on cell surface, while TLR3, TLR7, and TLR9 proteins are located both on the cell membrane and intracellularly. The highest expression was observed for TLR5 and the lowest for surface TLR7. These observations undoubtedly indicate that mature tissue mast cells have a broad set of TLR molecules, thus can recognize and bind bacterial, viral, and fungal PAMPs as well as various endogenous molecules generated in response to infection.

Endogenous antimicrobial factors in the treatment of infectious diseases.

Nowadays, a number of antibiotic-resistant bacteria strains is increasing. It is a serious clinical problem and poses a threat to the effectiveness of conventional antibiotic therapy. Thus, scientists are constantly seeking new alternatives for treatment of infectious diseases. There are some natural endogenous factors, which possess antimicrobial activities against a large number of microorganisms, including both Gram-positive and Gram-negative bacteria, viruses and fungi. These factors are present in all eukaryotic organisms and constitute an essential element of their immune system. A large number of in vitro and in vivo models have been used to show the activity of antimicrobial factors, and only few studies have been conducted on people. Results indicate that administration of these molecules is therapeutically beneficial. This review summarizes knowledge of selected endogenous antimicrobial agents, such as cathelicidins, defensins, histatins, lysozyme and lactoferrin. We also discuss potential uses of these factors in the treatment of infectious diseases.