MicroRNA hsa-let-7a facilitates staphylococcal small colony variants survival in the THP-1 macrophages by reshaping inflammatory responses.

Recent studies have provided emerging evidence of the critical involvement of microRNAs in host immune defence against bacterial infection and that likewise the expression of the miRNAs is profoundly impacted by a variety of pathogens to subvert the immune response. Here, we report the role of hsa-let-7a miRNA in response to Staphylococcus epidermidis Small Colony Variants infection. We also assessed whether the expression levels of inflammatory cytokines associated with the hsa-let-7a are manipulated by the pathogen and the effect of the IFN-γ priming on the expression of hsa-let-7a and the fate of SCVs/WTs in infected macrophages. A striking observation was the downregulation of the let-7a miRNA upon challenge of the THP-1 activated cells with the SCV isolates while no significant changes in expression were noticed after the infection of macrophages with their WT counterparts. Staphylococcus epidermidis WT and SCV strains were found to invade and survive in macrophages. A significant reduction in bacterial load for both phenotypes was observed in macrophages treated with let-7a mimic compared to untreated ones. Survival of WTs was augmented in cells treated with the inhibitor in 4 out of 5 strains as compared to the number of bacteria recovered from non-transfected cells. At the same time, let-7a inhibitor did not influence on the survival of SCVs in macrophages as their number was comparable to number recovered from non-transfected cells. When the ratio of both let-7a cytokine targets was compared, anti-inflammatory IL-10 cytokine was induced by SCVs predominantly, while the macrophage challenge with WTs was characterized by the inflammatory cytokine profile with high IL-6 and low IL-10 production. Moreover, the balance between pro-inflammatory and anti-inflammatory cytokines has been expectedly retrieved when macrophages were transfected with let-7a mimic before infection with WT or SCV strains. The results also show that IFN-γ likely regulates the macrophage environment contributing to the inflammatory response and elimination of bacteria from intracellular milieu by augmenting the synthesis of pro-inflammatory cytokines and supressing the anti-inflammatory IL-10. Our work has shown that SCVs have the potential to regulate the let-7a miRNA to balance the pro-inflammatory IL-6 with anti-inflammatory IL-10 and this mechanism is one of the ways in a complex regulatory network adopted by SCVs to promote their survival.

Antibacterial properties of Allium sativum L. against the most emerging multidrug-resistant bacteria and its synergy with antibiotics.

Garlic has long been known as the most effective plant species in treatment of bacterial infections. Considering the vast potential of garlic as a source of antimicrobial drugs, this study is aimed to evaluate the antibacterial activity of Allium sativum extracts and their interactions with selected antibiotics against drug-sensitive and multidrug-resistant isolates of emerging bacterial pathogens that are frequently found in healthcare settings. As shown by the in vitro data obtained in this study, the whole Allium sativum extract inhibited the growth of a broad range of bacteria, including multidrug-resistant strains with bactericidal or bacteriostatic effects. Depending on the organism, the susceptibility to fresh garlic extract was comparable to the conventional antibiotic gentamycin. Since the combinations of fresh garlic extract with gentamycin and ciprofloxacin inhibited both the drug sensitive and MDR bacteria, in most cases showing a synergistic or insignificant relationship, the potential use of such combinations may be beneficial, especially in inhibiting drug-resistant pathogens. The study results indicate the possibility of using garlic as e.g. a supplement used during antibiotic therapy, which may increase the effectiveness of gentamicin and ciprofloxacin.

The road to success of coagulase-negative staphylococci: clinical significance of small colony variants and their pathogenic role in persistent infections.

Bacterial small colony variants represent an important aspect of bacterial variability. They are naturally occurring microbial subpopulations with distinctive phenotypic and pathogenic traits, reported for many clinically important bacteria. In clinical terms, SCVs tend to be associated with persistence in host cells and tissues and are less susceptible to antibiotics than their wild-type (WT) counterparts. The increased tendency of SCVs to reside intracellularly where they are protected against the host immune responses and antimicrobial drugs is one of the crucial aspects linking SCVs to recurrent or chronic infections, which are difficult to treat. An important aspect of the SCV ability to persist in the host is the quiescent metabolic state, reduced immune response and expression a changed pattern of virulence factors, including a reduced expression of exotoxins and an increased expression of adhesins facilitating host cell uptake. The purpose of this review is to describe in greater detail the currently available data regarding CoNS SCV and, in particular, their clinical significance and possible mechanisms by which SCVs contribute to the pathogenesis of the chronic infections. It should be emphasized that in spite of an increasing clinical significance of this group of staphylococci, the number of studies unraveling the mechanisms of CoNS SCVs formation and their impact on the course of the infectious process is still scarce, lagging behind the studies on S. aureus SCVs.

Evaluation of the role of staphylococci in the pathomechanism of conjunctivitis.

PURPOSE: Determination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity. METHODS: The study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtitre plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay. RESULTS: The ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. About 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. About 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE. CONCLUSIONS: Adherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

The role of the PI3K/mTOR signaling pathway in Staphylococcus epidermidis small colony variants intracellular survival.

The objective of this study was to analyze how Staphylococcus epidermidis SCV and WT strains manipulate the PI3K/Akt/mTOR signaling pathway. Six S. epidermidis strains with normal phenotype (WT) and six S. epidermidis strains with SCV phenotype were isolated in parallel from six patients with the prosthetic hip joint infections. THP-1 activated cells were incubated with or without PI3K inhibitor-wortmannin or with mTOR inhibitor-rapamycin. Next, macrophages were exposed to S. epidermidis WT and SCV strains. After 4 h incubation, bacterial survival inside macrophages as well as PI3K-mTOR activation was analyzed. SCV strains of S. epidermidis increased the level of Akt phosphorylation, compared to uninfected macrophages and to their parental WT forms. Wild type variants of S. epidermidis phosphorylated Akt at similar or lower levels as control uninfected cells. Next, the induction of mTOR target, phosphorylated ribosomal protein S6, was measured in bacteria-infected macrophages. The level of phosphorylation was significantly reduced when the cells were exposed to WT strains of S. epidermidis. In contrast, the SCV strains activated S6 protein mostly at a level comparable to the control cells. Rapamycin inhibited mTOR activation as the number of p-S6 positive cells decreased in the tested cases. To conclude, the SCV strains activate the PI3K-Akt signaling pathway in opposite to WT strains. This fact however did not influence the increase in the number of live SCV bacteria as compared to the WT strains. Knowing that the PI3K-Akt pathway is involved in proinflammatory cytokines suppression, SCVs seem to use this pathway to reduce the inflammatory response during the infection.

A Novel Approach to Study the Effect of Ciprofloxacin on Biofilms of Corynebacterium spp. Using Confocal Laser Scanning Microscopy.

Non-diphtherial corynebacteria are Gram-positive rods that cause opportunistic infections, what is supported by their ability to produce biofilm on artificial surfaces. In this study, the characteristic of the biofilm produced on vascular and urological catheters was determined using a confocal microscopy for the most frequently involved in infections diphtheroid species. They were represented by the reference strains of Corynebacterium striatum ATCC 6940 and C. amycolatum ATCC 700207. The effect of ciprofloxacin on the biofilm produced by the antibiotic-susceptible C. striatum strain was evaluated using three concentrations of the antimicrobial agent (2 ×, 4 ×, and 6 × the MIC - the Minimum Inhibitory Concentration). The basis for the interpretation of results was the statistical analysis of maximum points readings from the surface comprising a total of 245 areas of the biofilm image under the confocal microscope. It was observed that ciprofloxacin at a concentration equal to 4 × MIC paradoxically caused an enlargement of areas with live bacteria within the biofilm. Biofilm destruction required the application of ciprofloxacin at a concentration higher than 6 × MIC. This suggests that the use of relatively low doses of antimicrobial agents may increase the number of live bacteria within the biofilm, and further facilitate their detachment from the biofilm's structure thus leading to the spread of bacteria into the bloodstream or to the neighboring tissues. The method of biofilm analysis presented here provides the original and novel approach to the investigation of the diphtheroid biofilms and their interaction with antimicrobial agents.Non-diphtherial corynebacteria are Gram-positive rods that cause opportunistic infections, what is supported by their ability to produce biofilm on artificial surfaces. In this study, the characteristic of the biofilm produced on vascular and urological catheters was determined using a confocal microscopy for the most frequently involved in infections diphtheroid species. They were represented by the reference strains of Corynebacterium striatum ATCC 6940 and C. amycolatum ATCC 700207. The effect of ciprofloxacin on the biofilm produced by the antibiotic-susceptible C. striatum strain was evaluated using three concentrations of the antimicrobial agent (2 ×, 4 ×, and 6 × the MIC ­ the Minimum Inhibitory Concentration). The basis for the interpretation of results was the statistical analysis of maximum points readings from the surface comprising a total of 245 areas of the biofilm image under the confocal microscope. It was observed that ciprofloxacin at a concentration equal to 4 × MIC paradoxically caused an enlargement of areas with live bacteria within the biofilm. Biofilm destruction required the application of ciprofloxacin at a concentration higher than 6 × MIC. This suggests that the use of relatively low doses of antimicrobial agents may increase the number of live bacteria within the biofilm, and further facilitate their detachment from the biofilm's structure thus leading to the spread of bacteria into the bloodstream or to the neighboring tissues. The method of biofilm analysis presented here provides the original and novel approach to the investigation of the diphtheroid biofilms and their interaction with antimicrobial agents.

Apoptosis Regulation in Dental Pulp Cells and PD-1/PD-L1 Expression Dynamics Under Ozone Exposure - A Pilot Approach.

This study aimed to determine the effect of ozone on the expression of Bax and Bcl-2 genes in dental pulp cells. Additionally, the programmed cell death protein 1, programmed death-ligand 1, and CD200 antigens were determined in lymphocytes to assess their surface expression. Dental pulp cells were cultured from extracted healthy third molars and characterized as dental pulp stromal cells. Gene expression of Bcl-2 and Bax was analyzed at 0 s, 6 s, and 12 s of ozone exposure using real-time PCR. Lymphocytes from dental pulp were subjected to ozone exposure for 12 s and PD-1, PD-L1, and CD200/CD200R expression was analyzed by flow cytometry. Upon exposure to ozone for 6 s, the Bcl-2 expression decreased significantly to -0.09, and at 12 s, it increased significantly to 0.3. Bax gene expression level increased significantly to 0.188 after 6 s exposure, and at 12 s, to 0.16. Lymphocytes exposed to ozone for 12 s showed minimal changes in PD-1, PD-L1, and CD200/CD200R expression levels, indicating that oxidative stress does not impact the signaling pathways regulating these molecules. The significant upregulation of Bcl-2 at 12 s highlights the cells' effort to protect themselves from prolonged oxidative stress, possibly tipping the balance toward cell survival and tissue repair. However, the absence of changes in PD-1 and PD-L1 expression on lymphocytes under oxidative stress suggests that these molecules are not sensitive to oxidative stress in this context.

In Vitro Effects of Silver Nanoparticles on Pathogenic Bacteria and on Metabolic Activity and Viability of Human Mesenchymal Stem Cells.

The rapid development of nanotechnology has led to the use of silver nanoparticles (Ag-NPs) in various biomedical fields. However, the effect of Ag-NPs on human mesenchymal stem cells (hMSCs) is not fully understood. Moreover, too frequent an exposure to products containing nanosilver in sublethal amounts raises widespread concerns that it will lead to the development of silver-resistant microorganisms. Therefore, this study aimed to evaluate the mechanism of action of Ag-NPs on hMSCs by analyzing the cellular uptake of Ag-NPs by the cells and its effect on their viability and to assess antimicrobial activity of Ag-NPs against emerging bacterial strains, including multidrug-resistant pathogens. For metabolic activity and viability evaluation, hMSCs were incubated with different concentrations of Ag-NPs (14 µg/mL, 7 µg/mL, and 3.5 µg/mL) for 10 min., 1 h and 24 h and subsequently analyzed for their viability by live-dead staining and metabolic activity by the MTS assay. The effect of Ag-NPs on bacterial pathogens was studied by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In conclusion, it was observed that exposure of hMSCs to Ag-NPs of size <10 nm has no cytotoxic effect on the metabolic activity of the cells at the concentration of 3.5 µg/mL, with minimal cytotoxic effect being observed at the concentration of 14 µg/mL after 24 h of incubation. Our findings also confirmed that Ag-NPs at the concentration of 4 µg/mL are effective broad-spectrum bactericidal agents, regardless of the antibiotic-resistance mechanism present in bacteria.

Evaluation of high-mobility group box 1 protein concentration in serum of patients with M. tuberculosis infection.

Infection with Mycobacterium tuberculosis is accompanied by an intense inflammatory response. Recently, a new mediator of inflammation, HMGB1 protein has been identified that contributes to acute lung injury. However, its role in the systemic inflammatory response in tuberculosis has not been thoroughly investigated. We investigated the systemic levels of HMGB1 and TNF-α in patients with active and latent lung tuberculosis as a prognostic marker of disease activity. The study was performed to 70 patients with confirmed Mycobacterium tuberculosis infection and other than tuberculosis lung diseases and in 20 healthy persons. Serum HMGB1 and TNF-α concentrations were measured by ELISA. The highest concentration of HMGB1 was detected in the bloodstream of people with Mtb infection (latent and active). Its concentration increased significantly in sera of patients with active tuberculosis (47.5 ng/ml), compared to patients with other lung diseases (36.87 ng/ml). TNF-α had significantly higher concentration in a patients group compared to healthy controls, with the highest concentration in the LTBI group of patients (0.136 ng/ml). We observed a strong positive correlation between TNF-α and HMGB1 concentrations in patients with tuberculosis infections. We conclude that HMGB1 is secreted during active and latent tuberculosis in the highest amounts compared to other lung diseases.

Assessment of cytokine release after in vitro stimulation of whole blood with legionella pneumophila in immunocompromised patients.

This study we examined ex vivo potential of the immune response after stimulation of whole blood with L. pneumophila SG 1, SG 2-14 and L. pneumophila standard strain ATCC 33152 in immunocompromised patients, such as: hemodialysis patients and patients after renal transplantation. The levels of TNF-α and IFN-γ in supernatants were measured with the use of commercial ELISA kits. The synthesis of TNF-α and IFN-γ after stimulation with L. pneumophila were analyzed in two aspects: differentiated stimulatory activity in relation to SG 1, SG 2-14 and ATCC 33152 L. pneumophila and differentiated response of the hemodialysis patients and patients after renal transplantation in relation to the control group. The positive and negative results of anti-L. pneumophila antibodies of two groups of our patients were found for the analysis of the stimulatory activity of L.pneumophila as a primary or secondary response. In patients with immunosuppression the response in the secretion of cytokines (TNF-α and IFN-γ) was reduced after stimulation of L. pneumophila SG 1 but in varying degrees after stimulation of L. pneumophila SG 2-14, which indicates that the risk of the infection is varied.

Postbiotic Fractions of Probiotics Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG Show Immune-Modulating Effects.

Probiotic bacteria belonging to Lactobacillus spp. are important producers of bioactive molecules, known as postbiotics, that play essential roles in the immunological support of the intestinal mucosa. In this study, the system of co-culture of intestinal epithelial cells with macrophage cells in vitro was used to study the potential effect of postbiotic fractions of L. rhamonosus and L. plantarum on the modulation of the immune response induced by pro-inflammatory stimuli. This study's results revealed that the presence of probiotic bacterial components on the mucosal surface in the early and late stage of inflammatory conditions is based on cellular interactions that control inflammation and consequent damage to the intestinal epithelium. In our studies, heat killed fractions of probiotic bacteria and their extracted proteins showed a beneficial effect on controlling inflammation, regardless of the strain tested, consequently protecting intestinal barrier damage. In conclusion, the presented results emphasize that the fractions of probiotic bacteria of L. plantarum and L. rhamnosus may play a significant role in the regulation of LPS-mediated cytotoxic activity in intestinal epithelial cells. The fractions of probiotic strains of L. rhamnosus and L. plantarum showed the potential to suppress inflammation, effectively activating the anti-inflammatory cytokine IL-10 and modulating the IL-18-related response.

Microbiota: A Missing Link in The Pathogenesis of Chronic Lung Inflammatory Diseases.

Chronic respiratory diseases account for high morbidity and mortality, with asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) being the most prevalent globally. Even though the diseases increase in prevalence, the exact underlying mechanisms have still not been fully understood. Despite their differences in nature, pathophysiologies, and clinical phenotypes, a growing body of evidence indicates that the presence of lung microbiota can shape the pathogenic processes underlying chronic inflammation, typically observed in the course of the diseases. Therefore, the characterization of the lung microbiota may shed new light on the pathogenesis of these diseases. Specifically, in chronic respiratory tract diseases, the human microbiota may contribute to the disease's development and severity. The present review explores the role of the microbiota in the area of chronic pulmonary diseases, especially COPD, asthma, and CF.

Mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates from Eastern Poland.

This study investigated mutations in the rpoB gene of rifampin-resistant isolates obtained from patients living in Eastern Poland. A total of 37 phenotypically and/or genotypically confirmed M. tuberculosis rifampin-resistant clinical isolates were included in this study. The strains were selected from symptomatic patients with a diagnosis of pulmonary and extrapulmonary tuberculosis. A line probe assay kit (INNO-LiPA rif Tb) was used for any specific mutational pattern of rpoB gene. Our data support the common notion that rifampin resistance genotypes with mutation at a critical codon, i.e. the one encoding Ser-531, is frequent in M. tuberculosis populations regardless of geographic origin. Our findings also suggest that in a geographic area such as Eastern Poland less common mutations of the rpoB gene occur more frequently. The frequency of substitution at codon 526 (His-Asp) was found to be high in Lublin. This study indicates that mutations associated with nucleotide replacements in codons 526 (His-Asp) and 531 (Ser-Leu) were associated with a high percentage of RMP resistance, whereas mutations in codons 516 (Asp-Val) and 526 (His-Tyr) were observed in a low percentage of RMP-resistance.

Cytokine Levels in the In Vitro Response of T Cells to Planktonic and Biofilm Corynebacterium amycolatum.

Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1ß, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1ß, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1ß, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1ß, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1ß, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1ß, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.

Monitoring Legionella species in hospital water systems. Link with disease and evaluation of different detection methods.

The aim of this work was to evaluate three currently available isolation methods for Legionella using water samples and swabs of a single pediatric hospital water system. Additionally, high risk patients were screened for the presence of Legionella pneumophila antigen in urine. Fifteen water samples and 11 swab samples were collected from distal sites at 18 sampling locations. The International Standard Method (PN-ISO11731-2) based on membrane filtration and direct culture of bacteria on selective media were compared with amoebic co-culture. The numbers of legionellae detected exceeded 10(2) cfu/100 ml in 50% of the samples. All the positive samples contained L. pneumophila SGs 2-14. Urine samples were obtained from 57 immunosuppressed children and screened for the presence of L. pneumophila serogroup (SG) 1 antigen by Legionella urinary antigen EIA. Of the 57 urine samples tested for the presence of Legionella pneumophila SG 1 antigen, none were positive. Our results highlight the value of combined membrane filtration and amoebic co-culture methods in detecting viable L. pneumophila strains. Direct plating of 0.2 ml water is a useful screening method for samples containing large bacterial amount.

Intraphagolysosomal conditions predispose to Staphylococcus epidermidis small colony variants persistence in macrophages.

Staphylococcus epidermidis small colony variants can survive inside macrophages and their survival has been proposed as a pivotal process in the pathogenesis of biomaterial associated infections. In the present study the intracellular location of clinical isolates of SCV and parental wild type strains inside macrophages was determined. Furthermore, the effect of IFN-γ and rapamycin on the level of SCV/WT as well as lysosomes colocalisation and iNOS induction in THP-activated macrophages in response to WT and SCV strains of Staphylococcus epidermidis were examined. It was demonstrated that SCV strain of S. epidermidis can survive and persist inside macrophages and its intracellular survival is supported by the induction of phagosomal acidification. The ability to reduce the high proportion of LysoTracker positive SCV containing phagosomes was exclusively found when IFN-γ was used. The findings suggest that IFN-γ mediates SCV killing via two distinct mechanisms, phagosome alkalisation and an increased iNOS synthesis, so the cytokine may control S. epidermidis WT and SCV infection in macrophages. Staphylococcus epidermidis SCV is a less potent stimulus of iNOS than the WT strain and the feature may help SCV to persist in hostile environment of macrophages. Rapamycin treatment did not influence the iNOS synthesis but reduced the percentage of both bacterial strains within acidic organelles. However, the percentage of SCV within LysoTracker positive organelles, even though reduced comparing to non-primed cells, was higher than in the WT strain indicating that Staphylococcus epidermidis possesses unique metabolic features allowing SCV to survive within macrophages.

Immunological parameters of candidiasis in cancer patients.

This study attempts to correlate the frequency of infection by Candida with the following immunological factors presented in cancer patients undergoing chemotherapy: neutrophil number; level of exogenous myeloperoxidase (MPO); cytokines IL-12, IL-10, TNF-alpha and IFN-gamma. A total of 31 serum and blood samples were tested for MPO and cytokines concentrations by using the ELISA technique. The level of cytokines and MPO was measured in serum samples and whole blood cell culture (WBC) supernates. We observed that: 1) MPO deficiency seems to be an important risk factor for invasive candidiasis independently of the neutrophil count; 2) the levels of cytokines in serum showed disturbances in cancer-patient group; 3) in vitro production of TNF-alpha, IFN-gamma and IL-10 by WBC is stimulated in cancer patients and to various degrees may be connected with previous Candida colonization or infection. In addition, production of IL-12 is decreased in cancer patients, and Candida spp. Occurrence may have significant influence on its blood concentration.

The prognostic and diagnostic markers of invasive candidiasis in patients during chemotherapy.

The aim of the work was the early detection of Candida spp. in clinical samples of patients with carcinoma ovariorum undergoing chemotherapy by comparing three indicators of candidiasis: presence of mannan and yeast DNA in the bloodstream and colonization of mucosal membranes by Candida species as a prognostic marker of deep candidiasis. Thirty-one women with carcinoma ovariorum, during chemotherapy without symptoms of deep fungal infections, were examined twice over a six-day period. C. albicans was the dominant organism isolated from mucosal membranes. Two serum samples were positive for mannan on the first day of examination. All these patients were previously colonized by Candida spp. on mucous membranes. Four patients were positive on the last day of examination. Three of these patients were colonized by Candida spp. C. albicans infection was detected early in 4 out of 12 clinical samples by a combination of PCR and mannan-detecting methods. Colonization increases the risk of deep candidiasis. PCR and antigen detection are fast and reliable methods for early detection of Candida in bloodstream. For patients at risk, the clinical samples must be tested by at least two independent methods.

[Change of interleukin-4 and interleukin-12 levels after therapy of multiple sclerosis relapse with methylprednisolone]. / Zmiany stezen interleukiny-4 i interleukiny-12 po leczeniu rzutu stwardnienia rozsianego metyloprednizolonem.

BACKGROUND AND PURPOSE: In the pathomechanism of multiple sclerosis (MS), a vital role is attributed to autoimmune responses. Higher activity of the disease is connected with an increased activity of proinflammatory cytokines while in remissions anti-inflammatory cytokines dominate. The aim of the study was to evaluate the effects of methylprednisolone treatment on the level of IL-4 and IL-12 and to determine whether their levels are related to the degree of disability and may be prognostic factors in the assessment of relapse sequelae. MATERIAL AND METHODS: The study included 32 patients with MS (according to McDonald's criteria) with relapses and remissions (17 patients with relapse who received methylprednisolone in the dose of 1.0 g i. v. on 5 consecutive days and 15 patients in remission). The control group consisted of 15 patients with non-inflammatory diseases of the nervous system. The levels of cytokines were determined by ELISA method using the Pharmingen kits. The examinations of patients in relapse were conducted before and after steroid therapy; the remaining patients were examined only once. RESULTS: In the relapse a visible increase in serum IL-12 level (p<0.05) was observed; its level after methylprednisolone treatment was found to be significantly decreased (p<0.001). The level of IL-4 was not significantly affected by steroid therapy. There was no relation between the severity of disability and cytokine levels. CONCLUSIONS: The level of IL-12 increases in MS relapses and decreases after methylprednisolone therapy. The changes in IL-4 and IL-12 levels are the manifestations of inflammatory reactions connected with the relapse; however, they do not directly indicate the extent of damage to CNS. They cannot be treated as a prognostic factor allowing to anticipate the consequences of the relapse.