Ewa Brzezińska-Błaszczyk

Decreased proinflammatory cytokines in cervicovaginal fluid, as measured in midgestation, are associated with preterm delivery.

The main aim of this study was to investigate the relationship between selected proinflammatory cytokines [interleukin IL‐1 alpha (IL‐1α), IL‐1 beta (IL‐1β), IL‐6 and IL‐8] concentrations in cervicovaginal fluid, as measured in midgestation, and the risk of subsequent preterm delivery.

The association between maternal cervicovaginal proinflammatory cytokines concentrations during pregnancy and subsequent early-onset neonatal infection

Abstract Objective: The aim of this study was to investigate the relationship between the concentration of selected proinflammatory cytokines (IL-1α, IL-1β, IL-6 and IL-8) in cervicovaginal fluid, as measured in midgestation, and the risk of early-onset neonatal infection (EONI). Method: Cervicovaginal fluids were obtained from a cohort of 114 pregnant women at 22 to 34 weeks gestation. The samples were analyzed for the concentrations of selected proinflammatory cytokines using standard enzyme-linked immunosorbent assay technique (ELISA). Lower genital tract microbiology was diagnosed using Gram stain method according to Spiegels criteria and by culture. Results: Mean gestational age at the time of sampling was 29.0 weeks. Mean time between sampling and delivery was 9.3 (SD 4.7) weeks. Bacterial vaginosis (BV) was diagnosed in 27.2% of subjects and M. hominis and U. urealyticum in 22.8% and 26.3%, respectively. Out of 114 women examined, 20 (17.5%) delivered newborns with EONI. Median cervicovaginal concentrations of IL-1α, IL-1β, IL-6 and IL-8 did not differ between women who delivered newborns with EONI as compared to women who delivered newborns without EONI. Women with pathological lower genital tract microflora and low IL-8 concentration (below 25th percentile) during pregnancy presented a significant risk of delivering newborns with EONI (OR=4.9; 95% CI, 1.1–22.8). Subjects with pathological lower genital tract microflora and a low concentration of more than one cytokine had the highest risk of delivering a newborn with EONI, OR=16.2, 95% CI, 1.1–234.0. Conclusions: Cytokine measurement in cervicovaginal fluid in early gestation could be useful for predicting subsequent EONI only among pregnant women with lower genital tract infection. Maternal genital tract immune hyporesponsiveness as represented by low concentrations of proinflammatory cytokines may create a permissive environment for ascending infection and may lead to subsequent EONI.

Serum levels of peptide cathelicidin LL-37 in elderly patients with depression

Cathelicidin LL-37 is a small cationic that plays an important role in antimicrobial defense, as it kills a broad spectrum of infectious agents by disrupting their membranes, including gram-positive and gram-negative bacteria, some viruses and fungi; and it neutralizes activity of bacterial endotoxins. Moreover, cathelicidin LL-37 exerts proinflammatory effect, while numerous reports indicate the role of inflammation in the development of depression. The purpose of this study was to evaluate the circulating levels of cathelicidin LL-37 in elderly depressed patients. Thirty-nine elderly (age ≥ 60 years) women with major depressive disorder and thirty-eight non-depressed elderly (age ≥ 60 years) women were included into the study. The mean serum cathelicidin LL-37 concentration in patients with depression and in healthy subjects were 2.40 ± 3.00ng/mL and 1.17 ± 3.04ng/mL, respectively, and the difference was statistically significant. No significant differences between mean serum CRP level and WBC count in MDD patients and control group were documented. There were no correlations between LL-37 level and age, BMI, GDS score, CRP level or WBC count. It can be assumed that elevated serum LL-37 levels in depressed patients may reflect inflammatory activation associated with depression.

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.

Cathelicidin LL-37 Affects Surface and Intracellular Toll-Like Receptor Expression in Tissue Mast Cells

Undoubtedly, mast cells take part in host defense against microorganisms as they are numerous at the portal of infection, they release many proinflammatory and antimicrobial mediators, and they express pattern recognition receptors, such as TLRs. These receptors play a key role in recognition and binding molecules associated with microorganisms and molecules associated with damage. Cathelicidins exhibit direct antimicrobial activities against a broad spectrum of microbes by perturbing their cell membranes. Accumulating evidence suggests a role for these molecules in supporting cell activation. We examined the impact of human cathelicidin LL-37 on tissue mast cell TLR expression and distribution. Depending on context, we show that LL-37 stimulation resulted in minor to major effects on TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 expression. Confocal microscopy analysis showed that, upon stimulation, TLRs may translocate from the cell interior to the surface and conversely. FPR2 and EGFR inhibitors reduced the increase in expression of selected receptors. We also established that LL-37 acts as a powerful inducer of CCL3 and ROS generation. These results showed that in response to LL-37, mast cells enhance the capability to detect invading pathogens by modulation of TLR expression in what may be involved FPR2 or EGFR molecules.

The RLR/NLR expression and pro-inflammatory activity of tissue mast cells are regulated by cathelicidin LL-37 and defensin hBD-2

Considering the significance of mast cells (MCs) in the course of various physiological and pathological processes, and the pivotal role of endogenous molecules, i.e., cathelicidins and defensins as multifunctional modulators, the study examines the constitutive and cathelicidin LL-37/defensin hBD-2-induced expression of certain NLRs and RLRs, i.e., NOD1, NOD2, and RIG-I, in fully-mature tissue MCs, and the impact of LL-37 and hBD-2 on MC pro-inflammatory activity. All experiments were carried out in vitro on freshly-isolated peritoneal (P)MCs. qRT-PCR, western blotting, flow cytometry, and confocal microscopy were used to evaluate both constitutive and LL-37/hBD-2-induced expression of NOD1, NOD2, and RIG-I receptors. ROS was determined using H2DCFDA, and Boyden microchamber assay was used to define the migratory response. Standard techniques assessed histamine, cysLT, and chemokine generation. PMCs express NOD1, NOD2, and RIG-I constitutively. LL-37 and hBD-2 enhance the expression and induce translocation of the studied receptors and directly activate the pro-inflammatory and migratory responses of PMCs. Observations demonstrate that LL-37 and hBD-2 might augment MC capability and sensitivity to NLR and RLR ligands and strengthen the role of MCs in inflammation.

Circulating cathelicidin LL-37 level is increased in euthymic patients with bipolar disorder

More and more data seems to imply that immune mechanisms are involved in the pathomechanism of bipolar disorder (BD). However, the primary role of cathelicidin LL-37 is defense against pathogens, more and more data indicated that this peptide strongly modulates immune system functioning and contributes to immune pathology of chronic and inflammatory diseases. No data is available on the level of LL-37 in bipolar patients. The aim of the study was to examine the circulating levels of cathelicidin LL-37 in euthymic patients with BD. Forty patients with BD and fifty-nine healthy volunteers were enrolled into the study. Concentration of LL-37 in serum was assessed using immunoenzymatic test ELISA. The mean LL-37 concentration in bipolar patients and in healthy subjects were 4.60u202f±u202f7.65u202fng/mL and 1.92u202f±u202f2.89u202fng/mL, respectively, and the difference was statistically significant (pu202f=u202f0.035). Within the BD group LL-37 level was significantly higher in women than in men (pu202f=u202f0.045). The evaluation of serum LL-37 concentration during stable 8u202fweek treatment indicated that at baseline (T1) mean level of LL-37 was 5.82u202f±u202f10.59u202fng/mL; and after treatment (T2) was 4.33u202f±u202f5.87u202fng/mL; the difference between T1 and T2 was not significant. Elevated serum levels of LL-37 in bipolar patients may suggest the role of this peptide in the pathomechanism of BD.

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

ObjectiveThe 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 methodsExperiments were done in vitro on fully mature tissue rat mast cells isolated from the peritoneal cavity, and leptin was used at concentrations 0.001–100xa0ng/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.ResultsLeptin 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.ConclusionsOur observations clearly documented that leptin promotes the pro-inflammatory activity of mast cells, and it thereby engages these cells in the inflammatory processes.

An overview of mast cell pattern recognition receptors

BackgroundMast 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.FindingsParticularly, 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.ConclusionThis review will focus on the current knowledge of PRRs expressed within different types of MCs.

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; Pearsons 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; Students t test). Thus, it can be hypothesized that archaea may participate in the endodontic microbial community.