Karolina Rudnicka

Chitosan-based hydrogel implants enriched with calcium ions intended for peripheral nervous tissue regeneration.

A new method for fabrication of chitosan-based hydrogel implants intended for peripheral nervous tissue regeneration was developed. The method is based on an electrodeposition phenomenon from a solution of chitosan and organic acid. In order to increase the mechanical strength of the implant, the solution was enriched with hydroxyapatite. Hydroxyapatite served as a source of calcium ions too. The influence of the concentration of the polymer and the additive on chemical, mechanical as well as biological properties of the obtained implant was evaluated. The study showed great dependence of the initial solution composition mainly on the physicochemical properties of the resulting structure. Basic in vitro cytotoxic and pro-inflammatory assays showed biocompatibility of manufactured implants, therefore, animal experimentations may be considered.

Helicobacter pylori vs coronary heart disease - searching for connections.

In this review, we discussed the findings and concepts underlying the potential role of Helicobacter pylori (H. pylori) infections in the initiation, development or persistence of atherosclerosis and coronary heart disease (CHD). This Gram-negative bacterium was described by Marshall and Warren in 1984. The majority of infected subjects carries and transmits H. pylori with no symptoms; however, in some individuals these bacteria may cause peptic ulcers, and even gastric cancers. The widespread prevalence of H. pylori infections and the fact that frequently they remain asymptomatic may suggest that, similarly to intestinal microflora, H. pylori may deliver antigens that stimulate not only local, but also systemic inflammatory response. Recently, possible association between H. pylori infection and extragastric disorders has been suggested. Knowledge on the etiology of atherosclerosis together with current findings in the area of H. pylori infections constitute the background for the newly proposed hypothesis that those two processes may be related. Many research studies confirm the indirect association between the prevalence of H. pylori and the occurrence of CHD. According to majority of findings the involvement of H. pylori in this process is based on the chronic inflammation which might facilitate the CHD-related pathologies. It needs to be elucidated, if the infection initiate or just accelerate the formation of atheromatous plaque.

Helicobacter pylori antigens as potential modulators of lymphocytes' cytotoxic activity

Helicobacter pylori (H.p) colonizes human gastric mucosa and causes gastric and duodenal ulcer disease or gastric cancer. Various H.p compounds may modulate the host immune response in regards to tolerance of the infection or disease development. The aim of this study was to determine whether H.p lipopolysaccharide (LPS) and glycine acid extract antigens (GE) or E. coli LPS influence the cytotoxic activity of peripheral blood lymphocytes from H.p infected – H.p (+) or uninfected – H.p (−) individuals, in the presence or absence of exogenous interleukin (IL)12. Individual H.p status was defined by the urea breath test. Lymphocytes, stimulated or not with H.p, and control antigens, with or without IL‐12, were used as effector cells and epithelial HeLa cells as targets. The cytotoxicity of lymphocytes was expressed as the percentage of dead target cells unable to reduce tetrazolium salt. The supernatants from HeLa/lymphocyte cultures were used for detection of the cellular cytotoxicity markers granzyme B and caspase 8. The natural cytotoxic activity of lymphocytes from H.p (+) was less than that of H.p (−) donors. This may have been due to fewer natural killer cells of CD3−CD56+Nkp46+ phenotype in H.p (+) in comparison to H.p (−) subjects. H.p GE and standard E. coli LPS enhanced the cytotoxicity of lymphocytes towards target cells whereas H.p LPS downregulated this activity. The decrease in lymphocyte cytotoxicity in response to H.p LPS correlated with a lack of IL‐2 and IL‐12 production, inhibition of interferon‐γ production, and low IL‐10 secretion by mononuclear leukocytes. IL‐12 significantly enhanced the natural as well as H.p LPS and H.p GE driven cytotoxic capacity of lymphocytes. In conclusion, H.p LPS may negatively modulate natural cytotoxic activity and cytokine secretion by immunocompetent cells and thus be involved in the maintenance of infection and development of gastric pathologies.

Interaction of Helicobacter pylori with C-Type Lectin Dendritic Cell-Specific ICAM Grabbing Nonintegrin

In this study we asked whether Helicobacter pylori whole cells and lipopolysaccharide (LPS) utilize sugar moieties of Lewis (Le) antigenic determinants to interact with DC-SIGN (dendritic cell specific ICAM grabbing nonintegrin) receptor on dendritic cells (DCs). For this purpose the soluble DC-SIGN/Fc adhesion assay and the THP-1 leukemia cells with induced expression of DC-SIGN were used. We showed that the binding specificity of DC-SIGN with H. pylori LeX/Y positive whole cells and H. pylori LPS of LeX/Y type was fucose dependent, whereas in LeXY negative H. pylori strains and LPS preparations without Lewis determinants, this binding was galactose dependent. The binding of soluble synthetic LeX and LeY to the DC-SIGN-like receptor on THP-1 cells was also observed. In conclusion, the LeXY dependent as well as independent binding of H. pylori whole cells and H. pylori LPS to DC-SIGN was described. Moreover, we demonstrated that THP-1 cells may serve as an in vitro model for the assessment of H. pylori-DC-SIGN interactions mediated by LeX and LeY determinants.

Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

Detection of biosurfactants in Bacillus species: genes and products identification.

To screen environmental Bacillus strains for detection of genes encoding the enzymes involved in biosurfactant synthesis and to evaluate their products e.g. surfactin, iturin and fengycin.

IL-33 and IL-4 impair barrier functions of human vascular endothelium via different mechanisms

The vascular endothelium forms a barrier that controls flow of solutes and proteins and the entry of leukocytes into tissue. Injured tissue releases IL-33, which then alarms the immune system and attracts Th2 cells, thus increasing local concentration of IL-4. The aim of the study was to assess the influence of IL-33 and IL-4 on barrier functions of the human endothelium, expression of tight and adherent junction proteins, apoptosis and adhesive molecule surface expression in human endothelium in order to describe the mechanism of this effect. IL-33 and IL-4 decreased endothelial integrity and increased permeability. When added together, both cytokines lowered the endothelial integrity twice as much as used alone. This effect was accompanied by the down-regulation of occludin and VE-cadherin mRNA expression. Additionally, IL-4, but not IL-33, induced cell apoptosis. Both IL-33 and IL-4 showed the additive potency to down-regulate VE-cadherin mRNA expression. IL-33, unlike IL-4, increased the surface expression of ICAM-1, but not PECAM-1 in endothelial cells. Our results indicate that IL-33 may reversibly destabilize the endothelial barrier, thus accelerating the supply with immunomodulators and assisting leukocytes to reach wounded tissue. However, extended and less-controlled down-regulation of endothelial barrier, which may be a consequence of IL-33-initiated, but in fact IL-4-induced apoptosis of endothelial cells, may be deleterious and may eventually lead to the aggravation of inflammatory processes and the prolongation of tissue dysfunction.

Immunophenotype of peripheral blood natural killer cells and IL-10 serum levels in relation to Helicobacter pylori status.

Recent findings suggest that NK (Natural Killer) cells may directly modulate the antimicrobial immune responses. In this study, we performed immunophenotypic analysis of peripheral blood NK cells with regard to CD56, CD16, Nkp46, and CD25 markers, as well as IL‐10 levels quantification in the sera samples of asymptomatic, H. pylori (Hp)‐infected or uninfected individuals, and combined these results with our previous findings on lymphocyte cytotoxic activity. Twenty healthy volunteers [10 Hp(−);10 Hp(+)] were included in the study. The percentages of classic lymphocytes (CD3+) and NK cells (CD3−CD56+, CD3−Nkp46+, CD3−CD16+) with or without CD25 receptor were evaluated by fluorochrome‐conjugated monoclonal antibody staining and flow cytometry analysis. IL‐10 quantification was performed by enzyme‐linked immunosorbent assay‐ELISA. Our study showed elevated levels of IL‐10 and higher NK cell numbers of both CD3−CD56+CD25+ and CD3−Nkp46+CD25+ phenotypes, as well as CD3+CD25+ classic lymphocytes in Hp(+) compared with Hp(−) individuals. No differences between Hp(−) and Hp(+) individuals were found either in total number of classic lymphocytes or NK cell subtypes. Our data suggest that in Hp(+) donors, there is a domination of lymphocytes and NK cells co‐expressing CD25 marker, which might be influenced by the regulatory IL‐10. This phenomenon may be a result of H. pylori adaptation to a changing environment in vivo leading to a chronic infection and lack of severe gastric pathologies.

Helicobacter pylori-driven modulation of NK cell expansion, intracellular cytokine expression and cytotoxic activity

During Helicobacter pylori (Hp) infections, innate immune cells may be positively or negatively modulated by Hp compounds or by Hp-induced cytokines. We have shown previously that the natural cytotoxic activity of PBMC was lower in Hp-infected [Hp(+)] than Hp-uninfected individuals [Hp(−)]. Here, we asked whether the Hp-modulated cytotoxic amplitude is associated with changes in the number of NK cells, their activation or intracellular cytokine expression. Flow cytometry immunophenotyping of PBMC was performed with regard to the surface receptors CD3, CD56 and CD25, and intracellular cytokine expression of IL-2, IFN-γ and IL-10 after in vitro stimulation with Hp glycine acid extract (GE), Hp LPS or standard Escherichia coli LPS. Hp GE-driven enhancement of lymphocyte cytotoxic activity was associated with the expansion of CD3−CD56+CD25+ NK cells and the up-regulation of IFN-γ and/or IL-2 synthesis, up to the higher level in Hp(−) than in Hp(+), while Hp LPS-mediated decrease in lymphocyte cytotoxicity was accompanied by the lack of CD3−CD56+CD25+ NK propagation, the inhibition of pro-inflammatory cytokine expression and intense expansion of IL-10–producing NK cells. Thus, the cytotoxic and cytokine activities of NK cells were dependent on the type of antigenic challenge and the Hp status, that is, NK cells could be modulated positively by Hp GE Ags and negatively by Hp LPS.

Putative consequences of exposure to Helicobacter pylori infection in patients with coronary heart disease in terms of humoral immune response and inflammation

Introduction Pathogens, including Helicobacter pylori (Hp), have been suggested to contribute to the development of coronary heart disease (CHD), although the evidence still remains insufficient. The study was focused on the exposure of CHD patients to Hp and resulting anti-Hp heat shock protein B HspB antibody production in relation to the level of serum lipopolysaccharide binding protein (LBP) as a marker of inflammation. Material and methods One hundred seventy CHD patients and 58 non-CHD individuals participated in this study. Coronary angiography confirmed the atheromatic background of CHD. The panel of classical risk factors included: arterial hypertension, diabetes, total cholesterol, low-density lipoprotein (LDL)/high-density lipoprotein (HDL) cholesterol, triglycerides, obesity and nicotinism. The Hp status was estimated by 13C urea breath test and serology. Immunoblot and ELISA were used for screening the sera samples for anti-Hp HspB immunoglobulins (Igs) and LBP. Results Coronary heart disease patients were exposed to Hp more frequently than non-CHD individuals. This was associated with increased levels of specific anti-Hp IgG2 and IgA as well as total IgA. Hp infected CHD and non-CHD donors produced anti-Hp HspB IgG cross-reacting with human Hsp 60. In CHD patients the LBP level was significantly higher in comparison to non-CHD donors. This was related to the severity of the disease. Type I Hp strains stimulated higher LBP levels than less pathogenic type II isolates. Conclusions Lipopolysaccharide binding protein secreted in excess together with anti-Hp HspB, cross-reacting with human Hsp60, may increase the risk of vascular pathologies in Hp-exposed CHD patients.