Type of monocyte immunomagnetic separation affects the morphology of monocyte-derived dendritic cells, as investigated by scanning electron microscopy.

Dendritic cells (DCs) are increasingly being used for multiple applications and are useful tools for many immunotherapeutic strategies. The understanding of the possible impact of the DCs-generation methods on the biological capacities of these cells is therefore essential. Although the immunomagnetic separation is regarded as a fast and accurate method yielding cells with the high purity and efficiency, still little is known about its impact on the properties of the generated DCs. The aim of this study was to compare the morphology of the monocyte derived dendritic cells (MoDCs), generated from monocytes selected with anti-CD14 mAbs (positive separation) and treated with anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123, glycophorin A (negative separation), using laser scanning microscopy. We found that the type of the immunomagnetic separation method used strongly influences the shape and cell dimension of the MoDCs. We observed that the height of both immature and LPS-matured DCs generated from monocytes isolated by negative separation was significantly higher compared to the cells obtained by positive separation.

Interactions between an M. tuberculosis strain overexpressing mtrA and mononuclear phagocytes.

PURPOSE: It was previously shown that the bacterial two-component regulatory signal transduction (2CR) system MtrAB may be associated with the ability of M. tuberculosis (Mtb) to survive in macrophages. In the present work Mtb mutants: Rv-78 with overexpression of mtrA and Rv-129 with elevated level of phosphorylation-defective MtrA were used for further investigation of the potential influence of the MtrAB system on Mtb interaction with human monocytes. MATERIAL/METHODS: Flow cytometry was used to determine the expression of MHC class II molecules. The expression of genes for inducible nitric oxide synthase (iNOS) and cathepsin G was quantified by RT-PCR. The association of Mtb strains with Rab5 and Rab7 positive vacuoles was investigated applying confocal microscopy. IL-10 and IL-12 secretion by monocytes as well as the Mtb susceptibility to cathepsin G were investigated. RESULTS: Mutation-carried and wild type Mtb strains inhibited MHC class II expression on monocytes to a similar extent. Monocyte stimulation with mycobacteria led to the increased production of IL-10 but no detectable amounts of IL-12 or NO were observed. Expression of the gene for iNOS was not detected while that for cathepsin G was shown, however its intensity was not associated with MtrA mutation. Mtb mutant strains were more effectively enclosed in phagosomes containing the late endosome marker Rab7 as compared to the control. CONCLUSIONS: The results may confirm the importance of the MtrAB system in mycobacterial capacity for successful survival in phagocytes, especially in the context of high degree of colocalization of Mtb Rv-78 to mature phagosomes.

Helicobacter pylori lipopolysaccharide activity in human peripheral blood mononuclear leukocyte cultures.

Helicobacter pylori (H. pylori) have been recognized as a major cause of chronic gastritis, gastric and duodenal ulcers and gastric cancer. Macrophages are the targets of lipopolysaccharide (LPS), which is a constituent of the outer membrane of Gram-negative rods. In this study we focused on a potential role of macrophages in the proliferation of human peripheral blood mononuclear leukocytes (PBML) in the milieu of H. pylori LPS and standard E. coli LPS. First, we found that H. pylori and E. coli LPS induced proliferation of total PBML (tPBML) from 5 out 21 healthy blood donors (LPS responders). In the LPS milieu, tPBML from the majority of volunteers (LPS non-responders) showed a significant decrease in the [(3)H]-thymidine incorporation as compared to tPBML in medium alone. The decreased cell proliferation was associated with a diminished metabolic activity of non-adherent lymphocytes. Then, non-adherent lymphocytes were stimulated with autologous macrophages pulsed with bacterial LPS. Still, the lymphocytes from the non-responders did not proliferate in the cultures with LPS exposed macrophages. In the group of LPS responders, the macrophages pulsed with H. pylori LPS significantly reduced the proliferation of non-adherent lymphocytes. The possible mechanism regulating the responses of PBML to bacterial LPS with an implication for the outcome of H. pylori infections is discussed.

BAFF from bone marrow-derived mesenchymal stromal cells of rheumatoid arthritis patients improves their B-cell viability-supporting properties.

Mesenchymal stromal cells (MSCs) represent a unique cell type with anti-proliferative effects on activated T and B cells. Based on our observation of differences between rheumatoid arthritis and osteoarthritis bone marrow B cells we hypothesized that rheumatoid arthritis bone marrow MSCs may enhance B-cell survival. We aimed to compare the effect of rheumatoid arthritis and osteoarthritis bone marrow-derived MSCs (rheumatoid arthritis MSCs, osteoarthritis MSCs) on the survival of healthy donor purified B cells. Rheumatoid arthritis and osteoarthritis MSCs were isolated from patients undergoing hip replacement surgery, and cultured in vitro for 2-5 passages. Washed cells were co-cultured with CD20+ B cells for 30-90 hours. Cell survival was analysed using 7-amino-actinomycin D labelling by flow cytometry. Expression of mRNA and protein was determined by RT-PCR and flow cytomery. Co-culture with both rheumatoid arthritis MSCs and osteoarthritis MSCs significantly enhanced B-cell survival, the effect being more prominent in rheumatoid arthritis MSCs. Both types of MSCs displayed expression of B cell-activating factor mRNA and protein. Blocking B cell-activating factor signalling from MSCs by specific anti-B cell-activating factor and anti-B cell-activating factor receptor antibodies weakly reversed the effect of MSCs on B-cell survival mainly in rheumatoid arthritis MSCs. MSC interaction with B cells provides stimuli for B-cell survival and therefore may contribute to the pathogenesis of rheumatoid arthritis. MSC-derived factors other than B cell-activating factor are likely to contribute to this effect. This feature is more prominent in rheumatoid arthritis MSCs, possibly due to the B cell-activating factor.

Comparison of taurine chloramine and taurine bromamine effects on rheumatoid arthritis synoviocytes.

Fibroblast-like synoviocytes (FLS) participate in rheumatoid arthritis (RA) chronic synovitis by producing pro-inflammatory cytokines (IL-6, IL-8), growth factors (VEGF) and other inflammatory mediators (PGE2, NO). We have previously reported that Tau-Cl, generated by neutrophils, inhibits in vitro some of these pathogenic RA FLS functions. Taurine bromamine (Tau-Br) originates from eosinophils and neutrophils, and its immunoregulatory activities are poorly known. Therefore, we investigated the effects of Tau-Br on RA FLS functions and compared it to Tau-Cl anti-inflammatory action. When applied at noncytotoxic concentrations: (i) Tau-Br inhibited IL-6 and PGE2 production with potency similar to Tau-Cl (IC50 approximately 250 microM), (ii) Tau-Br failed to affect VEGF and IL-8 synthesis, while Tau-Cl exerted inhibitory effect (IC50 approximately 400 microM), (iii) none of these compounds affected NO generation and iNOS expression. Thus, Tau-Cl is more effective than Tau-Br in normalization of pro-inflammatory RA FLS functions.

Taurine chloramine inhibits proliferation of rheumatoid arthritis synoviocytes by triggering a p53-dependent pathway.

OBJECTIVE AND DESIGN: Taurine chloramine (Tau-Cl), originating from activated neutrophils, possesses antiinflammatory activities. Fibroblast-like synoviocytes (FLS) participate in the chronic synovitis and synovial membrane hyperplasia that are characteristic pathological features of rheumatoid arthritis (RA). The present study was conducted to investigate the mechanism of the Tau-Cl effect on the proliferation of these cells in culture. MATERIALS AND METHODS: FLS were stimulated in vitro with platelet derived growth factor (PDGF) alone or together with Tau-Cl. Cell proliferation was evaluated by counting the total and dividing cell numbers and by measurement of (3)H-thymidine incorporation. Expression of the key cell-cycle regulators was evaluated at the protein (Western blotting) and/or mRNA (RT-PCR) levels. RESULTS: Treatment of RA FLS with Tau-Cl (200-500 microM) resulted in an early nuclear accumulation of p53 tumor suppressor protein. Moreover, Tau-Cl inhibited PDGF-triggered cell proliferation (IC(50) value approximately 250-300 microM), accompanied by characteristic modulation of p53 transcriptional targets: down-regulation of proliferating cell nuclear antigen (PCNA) and survivin, and concomitant up-regulation of p21 mitotic inhibitor. CONCLUSION: We propose that Tau-Cl inhibits proliferation of RA FLS by triggering a p53-dependent cell-cycle arrest and conclude that this compound suppresses pathways in FLS that are known to contribute to the pathology of RA.

Expression of IL-15 and IL-15 receptor isoforms in select structures of human fetal brain.

IL-15, a key cytokine linking innate and acquired immunity, is expressed in many cell types and tissues. Recent data indicate constitutive expression of IL-15 in human neural cell lines and tissues. The aim of the present study was to examine the expression patterns of mRNA encoding IL-15 and IL-15 receptor alpha (IL-15Ralpha) isoforms in select structures of human fetal brain. We report that mRNA for IL-15 and IL-15Ralpha isoforms were expressed in all tested brain structures: cerebral cortex, cerebellum, hippocampus, and thalamus. However, the levels of IL-15 and IL-15Ralpha mRNA were higher in the hippocampus and cerebellum in comparison with cortex and thalamus. Moreover, higher levels of cytosol in comparison with membrane-bound IL-15 isoform were present in all brain structures. The constitutive, but distinct, expression of IL-15 and its receptors in select human fetal brain structures suggests that IL-15 plays a role in their development and physiology.

Detection of phospholipase C in nontuberculous mycobacteria and its possible role in hemolytic activity.

Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH and plcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, and M. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected in M. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

A potential double role of anti-Lewis X antibodies in Helicobacter pylori-associated gastroduodenal diseases.

In this study, we found Lewis X (Le(x)) determinants on 68% of Helicobacter pylori isolates from patients with chronic gastroduodenal diseases. Anti-Le(x) IgG were detected more frequently in the sera from dyspeptic children and adults (45 and 46%), with or without proved (culture) H. pylori infection, than in the sera from healthy individuals (14% and 25%). In contrast, the prevalence of anti-Le(x) IgM was higher in the groups of healthy individuals than in the groups of dyspeptic patients. Moreover, anti-Le(x) monoclonal antibody of IgM class enhanced the uptake of Le(x)(+) but not Le(x)(-) H. pylori isolates by phagocytes. In the sera from some dyspeptic patients, we detected Le(x)-anti-Le(x) IgG immune complexes (Le(x) ICs). There was a great difference between children and adults as regards the presence of Le(x) ICs. The immune complexes were found in the sera from nine out of 29 (27%) H. pylori-infected and three out of eight (37%) uninfected adult dyspeptic patients. In comparison, Le(x)-anti-Le(x) IgG ICs were detected only for two out of 18 (11%) H. pylori-infected children. Le(x) ICs were not found in the sera from healthy individuals. Our results suggest that anti-Le(x) IgM may play a protective role in H. pylori infections. In contrast, anti-Le(x) IgG and particularly Le(x)-anti-Le(x) IgG ICs might contribute to the pathogenesis of chronic H. pylori infections.

Characterization of Staphylococcus aureus strains isolated from cystic fibrosis patients by conventional and molecular typing.

The phenotypic and genotypic characteristics of Staphylococcus aureus strains isolated from respiratory tract of cystic fibrosis (CF) patients were investigated. Slime production, cell-surface hydrophobicity, type of capsular polysaccharide, profile of heteroresistance to methicillin and Sma I restriction profiles were evaluated. S. aureus CF strains have been shown to be heterogeneous in respect to several important features. All of them were slime producing with variation in colony morphology. High or moderate cell-surface hydrophobicity (CSH) was found for, respectively, 16.2% and 83.8% strains. Thirty strains were resistant to methicillin, 60% of them showed heteroresitance and 40% were homoresistant. It was found that 59.6% of strains produced capsular polysaccharides (CP) of 5 or 8 type. Among CP5/CP8 strains, CP8 was the predominant type (81.1%). Typing of 62 CF strains by macrorestriction analysis of chromosomal DNA revealed several major types, differing in their SmaI profiles with a similarity coefficient lower than 0.4. Some of the strains isolated from the same patient at different times of hospitalization, as well as strains isolated at the same time from the relatives, were identical in their PFGE pattern.

Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections.

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.

[Production of reactive nitrogen and oxygen intermediates in human granulocytes and monocytes during internalization of live BCG bacilli]. / Powstawanie aktywnych form azotu i tlenu w ludzkich granulocytach i monocytach pochlaniajacych zywe pratki BCG.

The production of nitric oxide (NO) and reactive oxygen intermediates in granulocytes and macrophages from healthy volunteers, infected in vitro with live Bacille Calmette-Guerin (BCG) mycobacteria, was estimated. Significant differences in the biochemical reactions induced by BCG bacilli in granulocytes and monocytes are described. The activity of phagocytes was also investigated in the cultures with cytokines: IFN-gamma, TNF-alpha, GM-CSF, IL-4.

The production of nitric oxide and tumor necrosis factor by murine macrophages infected with mycobacterial strains differing by hemolytic activity.

In this study, we compared the secretion of nitric oxide (NO) and tumor necrosis factor (TNF-alpha) by murine macrophages infected in vitro with hemolytic or unhemolytic mycobacteria isolates. We observed that unhemolytic mycobacteria induced more intensive NO production by macrophages and were more susceptible to bactericidal effect of mononuclear phagocytes than hemolytic mycobacterial strains. In contrast, the high-virulence hemolytic isolates induced significantly stronger TNF-alpha production by infected macrophages than the low-virulence unhemolytic bacilli.

Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods.

The aim of this work was to compare the possibility of identifying Listeria monocytogenes strains isolated from meat and sausage on the basis of the API-Listeria test, production of phosphatidylinositol-specific phospholipase C (PI-PLC) and polymerase chain reaction (PCR) for a DNA fragment of the hlyA gene encoding listeriolysin O. Forty-six strains were isolated and examined. The lethality of some Listeria isolates for BALB/c mice was also determined. In this study, all isolates identified as L. monocytogenes in the API test gave a positive signal in the PCR. Listeriae identified as L. innocua or L. welshimeri in the API test were negative in the PCR conducted with the primers for listeriolysin O. All strains identified as L. monocytogenes on the basis of the API test and the PCR produced PI-PLC. However, this activity was not limited to the bacteria of this species. Four out of 17 L. innocua and three out of 10 L. welshimeri isolates were PI-PLC-positive. None of the L. innocua or L. welshimeri isolates (neither PI-PLC+ or PI-PLC-) showed lethality for BALB/c mice. In contrast, two L. monocytogenes isolates as well as a reference L. monocytogenes strain killed all mice used for the experiment.

Anti-Lewis X IgM and IgG in H. pylori infections in children and adults.

A role of autoimmune processes in the pathology of Helicobacter pylori infections has been suggested. The Lewis determinants present in LPS molecule of H. pylori bacteria have been indicated as the cause of antigenic mimicry. In this study, the prevalence of IgM and IgG antibodies to Lewis X antigen in the sera from children and adults, with or without dyspepsia, infected or not infected with H. pylori, seropositive and seronegative for anti-H. pylori IgG were determined immuno-enzymatically (ELISA). Our results revealed that humans may produce anti-Lewis X antibodies, particularly of IgM class, in the absence of H. pylori infection or H. pylori independent dyspepsia. The production of such antibodies, by healthy children who had never been infected with H. pylori suggested that anti-Lewis X antibodies may occur naturally.

[IgG and IgA immunoglobulins in helicobacter pylori infections of children with chronic dyspepsia before and after two week triple drug therapy]. / Immunoglobuliny IgG i IgA w zakazeniach Helicobacter pylori dzieci z przewlekla dyspepsja przed i po dwutygodniowej trójlekowej terapii.

The aim of this study was to investigate whether serological techniques, ELISA and Western blot, are useful in monitoring treatment of H. pylori-associated chronic dyspeptic symptoms in children. We observed a correlation between a decrease in the anti-H. pylori IgG titer and an effective treatment. So, our results suggested that the ELISA assay conducted with a glycine H. pylori extract can be a good noninvasive assay for monitoring the effectiveness of the therapy. By using the Western blot method, we showed some variation in the specificity of anti-H. pylori IgG produced before and after treatment. However, this variation was not correlated with the effectiveness of the therapy.

[Detection of bacterial biofilm on medical biomaterials]. / Wykrywanie biofilmu bakteryjnego na biomaterialach medycznych.

This study was performed to assess the value TTC assay in the diagnosis of biomaterial-associated infections. In this assay, soluble colourless TTC is reduced to insoluble red formazan by electron transfer associated with active oxidative bacterial metabolism and is precipitated intracellularly. Microbial adhesion and biofilm formation on the surface of medical prosthetic devices (vesicular and urinary catheters) made of various polymers (PTFE, H-PE, PCW, SL), were determined. The microorganisms which are most often isolated in medical device-associated infections: S. aureus, S. epidermidis, E. faecalis, E. coli, P. vulgaris, P. aeruginosa, C. albicans, were included into the study. The obtained results indicate that the assay using TTC as a metabolic indicator of bacterial biofilm presence, is technically simple to conduct with minimal setup time. Even when classical cultures yielded no bacterial growth, TTC assessments demonstrated bacterial biofilms. TTC assay could be recommended as a quick routine method for confirmation of biomaterial device-associated infection.

The virulence of Staphylococcus aureus isolates differing by siderophore production.

In previous study we demonstrated that Staphylococci aureus clinical and environmental isolates differ by siderophore production (Lisiecki et al., 1997), the aim of the present study was to check a possible role of siderophore-dependent iron acquisition system of outcome of staphylococcal diseases. The systemic and local staphylococcal infections were induced in mice by inoculation of three S. aureus strains differing by siderophore production. We found that S. aureus B 47 strain characterized by enhanced siderophore activity was more virulent in both systemic and local infection models and it was more resistant to anti-bacterial activity of neutrophils than S. aureus B 63 and B 32 strains expressing weaker siderophore production. The results suggest that effective siderophore-dependent iron acquisition system may be beneficial to S. aureus strains in their pathogenic activity in vivo.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-coated implants and their potential for reducing biomaterial-associated infection in neutropenic hosts.

The incidence of infections associated with the use of medical biomaterials is high for skin-penetrating devices, when microbes of the normal skin flora like coagulase-positive and coagulase-negative staphylococci dominate as causative organisms. The most serious ones are infections in immunocompromised individuals. A mouse model of subcutaneous staphylococcal infection yielding abscesses in cyclophosphamide-induced neutropenic mice implanted with heparinized polyethylene (H-PE) was used. The present study addresses the question of the effects of implant modification with recombinant granulocyte-macrophage stimulating factor (rGM-CSF) on the course of infection. Our findings demonstrate that such modification reduces the proliferation of bacteria within the abscess and as a consequence limits the dissemination of bacteria from the local infection induced in the neutropenic host.

Systemic humoral response to Helicobacter pylori in children and adults.

In this study we compared the development of anti-H. pylori humoral response in adults and children. Two antigens: H. pylori acid glycine extract (GE) and recombinant cagA were used for ELISA and Western blot. Anti-GE IgG were detected in all and anti-cagA IgG in about 50% of H. pylori infected adults and children. The prevalence of anti-GE and anti-cagA IgG in the sera from H. pylori-uninfected children with gastritis/gastroduodenitis was lower than in the sera from healthy adult blood donors. Serum IgA were demonstrated for 71% of H. pylori-infected adults and for a smaller proportion (about 30%) of uninfected adult patients or normal subjects. Such antibodies were detected neither for infected nor for uninfected children. There was an evident difference between the proteins of H. pylori glycine extract recognized by antibodies present in the sera from H. pylori-infected children and adults. The antigen of molecular weight over 107 kDa was recognized exclusively by the sera from 30% of H. pylori-infected adults. The 80-107 kDa bands were recognized more frequently by the sera from adults than from children. In contrast, sera from infected children more frequently than sera from infected adults reacted with the bands of 14 kDa, 19 kDa and 26 kDa. The H. pylori antigens recognized by IgG, produced by infected children and adult patients, should be taken into consideration in the developing of tests for serodiagnosis of H. pylori infection.