Saccharomyces cerevisiae ß-glucan improves the response of trained macrophages to severe P. aeruginosa infections.

OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with ß-glucan might improve their response to P. aeruginosa infections. METHODS: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae ß-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo ß-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared. RESULTS: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced. CONCLUSIONS: Training of murine macrophages with S. cerevisiae ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.

Biofilm-forming strains of P. aeruginosa and S. aureus isolated from cystic fibrosis patients differently affect inflammatory phenotype of macrophages.

OBJECTIVE: Lung cystic fibrosis (CF) is characterized by chronic infections and hyperinflammatory response of neutrophils and macrophages. P. aeruginosa (PA) and S. aureus (MSSA, MRSA) are major pathogens of advanced CF. The main goal of this study was to compare the inflammatory phenotype of murine C57BL/6 macrophages exposed to PA57 with that exposed to MSSA60, both strains isolated from the same patient with severe CF. In the present study, we used C57BL/6 mice sensitive to lung infection with P. aeruginosa. METHODS: We measured the release of cytokines and the expression of phenotypic markers of murine neutrophils and macrophages exposed to bacterial cells and biofilm components (i.e., EPS) of the selected bacteria. In addition, a quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. RESULTS: Neutrophils stimulated with PA57 and MSSA60 strains produced hyperinflammatory pattern of cytokines. The pro-inflammatory impact of PA57 was significantly higher than that of MSSA60 (IL-6/IL-10 ratio: PA57 = 9.3 vs. MSSA60 = 1.7). Macrophages produced significantly lower amount of cytokines, but showed classical pattern of M1 markers (iNOS-High; arginase-1 and mannose receptor MRC1-Low). Importantly, as evidenced by proteomic analysis, PA57 and PA57-EPS were stronger inducers of M1 macrophage polarization than the MSSA60 counterparts. CONCLUSIONS: Our study demonstrated that strong biofilm P. aeruginosa strains, CF isolates, are dominant inducers of M1 macrophages, termed biofilm-associated macrophages (BAMs). We suggest that repolarization of detrimental BAMs might be a new therapeutic strategy to ameliorate the airway damage in CF.

Anti-Inflammatory Activities of Captopril and Diuretics on Macrophage Activity in Mouse Humoral Immune Response.

Hypertension is accompanied by the over-activation of macrophages. Diuretics administered alone or in combination with hypotensive drugs may have immunomodulatory effects. Thus, the influence of tested drugs on mouse macrophage-mediated humoral immunity was investigated. Mice were treated intraperitoneally with captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) by 8 days. Mineral oil-induced peritoneal macrophages were harvested to assess the generation of cytokines in ELISA, and the expression of surface markers was analyzed cytometrically. Macrophages were also pulsed with sheep red blood cells (SRBC) and transferred to naive mice for evaluation of their ability to induce a humoral immune response. Tested drugs increase the expression of surface markers important for the antigen phagocytosis and presentation. SRBC-pulsed macrophages from mice treated with captopril combined with diuretics increased the secretion of antigen-specific antibodies by recipient B cells, while macrophages of mice treated with hydrochlorothiazide or furosemide with captopril increased the number of antigen-specific B cells. Tested drugs alter the macrophage secretory profile in favor of anti-inflammatory cytokines. Our results showed that diuretics with or without captopril modulate the humoral response by affecting the function of macrophages, which has significant translational potential in assessing the safety of antihypertensive therapy.

Chronic bacterial pulmonary infections in advanced cystic fibrosis differently affect the level of sputum neutrophil elastase, IL-8 and IL-6.

Advanced cystic fibrosis (CF) lung disease is commonly characterized by a chronic Pseudomonas aeruginosa infection and destructive inflammation caused by neutrophils. However, the lack of convincing evidence from most informative biomarkers of severe lung dysfunction (SLD-CF) has hampered the formulation of a conclusive, targeted diagnosis of CF. The aim of this study was to determine whether SLD-CF is related to the high concentration of sputum inflammatory mediators and the presence of biofilm-forming bacterial strains. Forty-one patients with advanced CF lung disease were studied. The severity of pulmonary dysfunction was defined by forced expiratory volume in 1 second (FEV1) < 40%. C-reactive protein (CRP) and NLR (neutrophil-lymphocyte ratio) were examined as representative blood-based markers of inflammation. Expectorated sputum was collected and analysed for cytokines and neutrophil-derived defence proteins. Isolated sputum bacteria were identified and their biofilm-forming capacity was determined. There was no association between FEV1% and total number of sputum bacteria. However, in the high biofilm-forming group the median FEV1 was < 40%. Importantly, high density of sputum bacteria was associated with increased concentrations of neutrophil elastase and interleukin (IL)-8 and low concentrations of IL-6 and IL-10. The low concentration of sputum IL-6 is unique for CF and distinct from that observed in other chronic pulmonary inflammatory diseases. These findings strongly suggest that expectorated sputum is an informative source of pulmonary biomarkers representative for advanced CF and may replace more invasive bronchoalveolar lavage analysis to monitor the disease. We recommend to use of the following inflammatory biomarkers: blood CRP, NLR and sputum elastase, IL-6, IL-8 and IL-10.

Captopril Combined with Furosemide or Hydrochlorothiazide Affects Macrophage Functions in Mouse Contact Hypersensitivity Response.

Hypertension is a chronic disease associated with chronic inflammation involving activated macrophages. Antihypertensive drugs (for example, angiotensin-converting enzyme inhibitors-ACEIs) used in the treatment of hypertension have immunomodulatory properties. On the other hand, the immunological effect of diuretics and combined drugs (diuretics + ACEI) is unclear. Therefore, we examined the influence of diuretics and combination drugs (ACEI + diuretic) on cellular response (contact hypersensitivity), production of reactive oxygen intermediates (ROIs), and nitric oxide (NO), and the secretion of interleukin-12 (IL-12). CBA mice were administered i.p. captopril (5 mg/kg) with or without hydrochlorothiazide (10 mg/kg) or furosemide (5 mg/kg) for 8 days. On the third day, the mice were administered i.p. mineral oil, and macrophages were collected 5 days later. In the presented results, we show that diuretics administered alone or with captopril increase the generation of ROIs and reduce the formation of NO by macrophages. Moreover, tested drugs inhibit the secretion of IL-12. Diuretics and combined drugs reduce the activity of contact hypersensitivity (both activation and induction phases). Our research shows that the tested drugs modulate the cellular response by influencing the function of macrophages, which is important in assessing the safety of antihypertensive therapy.

Combined Biological Effects of N-Bromotaurine Analogs and Ibuprofen. Part I: Influence on Inflammatory Properties of Macrophages.

Taurine haloamines (N-chlorotaurine, N-bromotaurine) due to their strong antiseptic and anti-inflammatory properties are good candidates for topical application in treatment of skin inflammatory/infectious disorders. Recently, we have demonstrated that more stable N-bromotaurine analogs (N-dibromo-dimethyl taurine, N-monobromo-dimethyl taurine) and bromamine T show strong microbicidal and anti-inflammatory properties at concentrations well tolerated by human cells and tissue. Non-steroidal anti-inflammatory drugs (NSAIDs) with cyclooxygenase (COX) inhibitory activity are commonly used in various inflammatory diseases. However, systemic administration of NSAIDs may result in adverse side effects. For example, the use of ibuprofen in children with varicella is associated with enhanced serum levels of TNF-α and with increased risk of necrotizing soft tissue infections and secondary skin infections caused by invasive streptococci. The aim of this study was to examine combined immunomodulatory effects of bromamines and ibuprofen on J774.A1 macrophages. We have shown that the primary activity of ibuprofen, the inhibition of PGE2 production by activated macrophages was intensified in the presence of bromamines. Most importantly, the stimulatory effect of ibuprofen on production of inflammatory cytokines (TNF-α, IL-6) was inhibited by all tested bromamines. These observations indicate that bromamines may neutralize massive production of TNF-α at sites of inflammation, a side effect of ibuprofen. Therefore, we suggest that systemic administration of ibuprofen (NSAIDs) in treatment of inflammatory/infectious skin diseases should be supported by topical application of bromamines as an adjunctive therapy.

Combined Biological Effects of N-Bromotaurine Analogs and Ibuprofen. Part II: Influence on a Local Defense System.

The stable N-bromotaurine analogs (N-dibromo-dimethyl taurine, N-monobromo-dimethyl taurine), and bromamine T (BAT) show anti-inflammatory and microbicidal properties. These bromamines are good candidates for a treatment of skin infectious/inflammatory diseases as local antiseptics. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), is commonly used in various infectious/inflammatory diseases due to its analgesic and antipyretic therapeutic effects. However, systemic administration of ibuprofen may also result in adverse side effects. It has been reported that ibuprofen enhances serum levels of TNF-α and worsens secondary skin infections caused by invasive streptococci (S. pyogenes). Recently we have demonstrated that bromamines inhibit the stimulatory effect of ibuprofen on the production of inflammatory cytokines (TNF-α, IL-6). The aim of this study was to examine the combined antibacterial actions of ibuprofen and bromamines against S. pyogenes and their joint effect on the generation of reactive oxygen species (ROS) by activated neutrophils and macrophages. We have shown that the microbicidal activity of bromamines against S. pyogenes was not altered by ibuprofen. On the other hand, co-administration of ibuprofen and bromamines markedly decreased the generation of ROS by activated neutrophils and macrophages. Finally, we discuss how the antioxidant combined effect of bromamines and ibuprofen may affect a local defense system.

Pseudomonas aeruginosa biofilm is a potent inducer of phagocyte hyperinflammation.

OBJECTIVE: Pseudomonas aeruginosa effectively facilitate resistance to phagocyte killing by biofilm formation. However, the cross talk between biofilm components and phagocytes is still unclear. We hypothesize that a biofilm provides a concentrated extracellular source of LPS, DNA and exopolysaccharides (EPS), which polarize neighbouring phagocytes into an adverse hyperinflammatory state of activation. METHODS: We measured the release of a panel of mediators produced in vitro by murine neutrophils and macrophages exposed to various biofilm components of P. aeruginosa cultures. RESULTS: We found that conditioned media from a high biofilm-producing strain of P. aeruginosa, PAR5, accumulated high concentrations of extracellular bacterial LPS, DNA and EPS by 72 h. These conditioned media induced phagocytes to release a hyperinflammatory pattern of mediators, with enhanced levels of TNF-α, IL-6, IL12p40, PGE2 and NO. Moreover, the phagocytes also upregulated COX-2 and iNOS with no influence on the expression of arginase-1. CONCLUSIONS: Phagocytes exposed to biofilm microenvironment, called by us biofilm-associated neutrophils/macrophages (BANs/BAMs), display secretory properties similar to that of N1/M1-type phagocytes. These results suggest that in vivo high concentrations of LPS and DNA, trapped in biofilm by EPS, might convert infiltrating phagocytes into cells responsible for tissue injury without direct contact with bacteria and phagocytosis.

Cellular Interactions in the Intestinal Stem Cell Niche.

Epithelial cells are one of the most actively cycling cells in a mammalian organism and therefore are prone to malignant transformation. Already during organogenesis, the connective tissue (mesenchyme) provides instructive signals for the epithelium. In an adult organism, the mesenchyme is believed to provide crucial regulatory signals for the maintenance and regeneration of epithelial cells. Here, we discuss the role of intestinal myofibroblasts, α-smooth muscle actin-positive stromal (mesenchymal) cells, as an important regulatory part of the intestinal stem cell niche. Better understanding of the cross-talk between myofibroblasts and the epithelium in the intestine has implications for advances in regenerative medicine, and improved therapeutic strategies for inflammatory bowel disease, intestinal fibrosis and colorectal cancer.

Phagocytosis of live versus killed or fluorescently labeled bacteria by macrophages differ in both magnitude and receptor specificity.

Scavenger receptor (SR)-mediated opsonin-independent phagocytosis of bacteria by macrophages has been suggested to represent an important, early mechanism of anti-bacterial host defense. However, although the ability to bind bacteria has been demonstrated to be a shared feature of all types of SRs, in many cases the evidence is limited to the demonstration of increased binding of killed, fluorescently labeled bacteria to non-phagocytic cells transfected with these receptors. We sought to verify the ability of SRs to mediate non-opsonic phagocytosis of live Escherichia coli (Ec) and Staphylococcus aureus (Sa), model species of Gram-negative and -positive bacteria, respectively, and to assess the relative contributions of different SRs expressed on murine macrophages in this process. We found that the class A SR SR-A/CD204 was the major receptor mediating phagocytosis of fluorescently labeled Sa, whereas different SRs had highly redundant roles in the phagocytosis of live Sa. Conversely, different SRs contributed to the phagocytosis of fluorescently labeled Ec. In comparison, phagocytosis of live Ec was of much lower magnitude and was selectively mediated by SR-A. These results question the use of fluorescently labeled bacteria as valid replacements for live bacteria. The low magnitude of opsonin-independent phagocytosis of Ec and unimpaired phagocytosis of Sa in SR-A- or CD36-deficient macrophages indicate that the defect in this process might not be responsible for the reported impaired bacteria clearance in mice deficient in these receptors. We postulate that this impairment might result to a larger extent from inhibition of intracellular bacteria killing caused by pro-inflammatory cytokines, produced in excessive amounts by SR-deficient cells in response to bacterial products.

The impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation.

Translocation of bacteria, primarily Gram-negative pathogenic flora, from the intestinal lumen into the circulatory system leads to sepsis. In newborns, and especially very low birth weight infants, sepsis is a major cause of morbidity and mortality. The results of recently conducted clinical trials suggest that lactoferrin, an iron-binding protein that is abundant in mammalian colostrum and milk, may be an effective agent in preventing sepsis in newborns. However, despite numerous basic studies on lactoferrin, very little is known about how metal saturation of this protein affects a host's health. Therefore, the main objective of this study was to elucidate how iron-depleted, iron-saturated, and manganese-saturated forms of lactoferrin regulate intestinal barrier function via interactions with epithelial cells and macrophages. For these studies, a human intestinal epithelial cell line, Caco-2, was used. In this model, none of the tested lactoferrin forms induced higher levels of apoptosis or necrosis. There was also no change in the production of tight junction proteins regardless of lactoferrin metal saturation status. None of the tested forms induced a pro-inflammatory response in Caco-2 cells or in macrophages either. However, the various lactoferrin forms did effectively inhibit the pro-inflammatory response in macrophages that were activated with lipopolysaccharide with the most potent effect observed for apolactoferrin. Lactoferrin that was not bound to its cognate receptor was able to bind and neutralize lipopolysaccharide. Lactoferrin was also able to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect. Therefore, we hypothesize that lactoferrin supplementation is a relevant strategy for preventing sepsis.

1-Methylnicotinamide protects against liver injury induced by concanavalin A via a prostacyclin-dependent mechanism: A possible involvement of IL-4 and TNF-α.

We have recently demonstrated that concanavalin A (Con A)-induced hepatitis is associated with the release of endogenous 1-methylnicotinamide (MNA). Here we study the mechanism by which exogenous MNA alleviates Con A-induced liver inflammation and injury in vivo. The involvement of prostacyclin (PGI2) in hepatoprotective action of MNA (30-100 mg kg(-1); i.v.) was studied by the use of IP receptor antagonist RO3244794 (10 mg kg(-1); p.o.) given prior to Con A (5-20 mg kg(-1); i.v.). Liver damage was assessed by measurements of: liver specific transaminases in plasma (alanine aminotransferase; aspartate aminotransferase); cytokines release (IL-4, IFN-γ and TNF-α); liver histopathology; and 24h survival rates. Additionally, the effect of a stable analog of prostacyclin (carbaprostacyclin) on IL-4, IFN-γ and TNF-α production by isolated spleen lymphocytes in response to Con A was analyzed. MNA diminished Con A-induced rise in liver specific transaminases, alleviated histopathological injury and improved 24h survival rates, the latter effect in a degree comparable with the pretreatment of animals with dexamethasone (0.5 mg kg(-1); i.p.). MNA inhibited also a rise in IL-4 and TNF-α concentration in plasma measured 2 h after Con A administration, while IFN-γ was less affected. The effects of MNA were reversed by pretreatment with IP antagonist RO3244794. In isolated spleen lymphocytes, carbaprostacyclin profoundly decreased production of IL-4, the effect on TNF-α was modest with no effect on IFN-γ production. In conclusion, MNA attenuated Con A-induced hepatitis by a prostacyclin-dependent mechanism involving the inhibition of lymphocytes-derived IL-4 and the inhibition of Kuppfer-cells derived TNF-α.

Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

Staphylococcus epidermidis and biofilm-associated neutrophils in chronic rhinosinusitis. A pilot study.

A key role of bacterial biofilm in the pathogenesis of chronic rhinosinusitis (CRS) with (CRSwNP) and without nasal polyps (CRSsNP) is commonly accepted. However, the impact of some bacterial species isolated from inflamed sinus mucosa on biofilm formation is unclear. In particular, the role of Staphylococcus epidermidis as aetiological agents of CRS is controversial. Moreover, the effect of biofilm formation on neutrophil infiltration and activity in CRSwNP calls for explanation. In this study, biofilms were found in three of 10 patients (mean age = 46 ± 14) with CRS undergoing endoscopic sinus surgery by means of scanning electron microscopy. Unexpectedly, S. epidermidis was the primary isolated bacteria and was also found to be present in all biofilm-positive mucosa specimens, indicating its pivotal role in the pathogenesis of severe chronic infections associated with biofilm formation. We have also measured the activity of myeloperoxidase (MPO), the most abundant neutrophil enzyme, to demonstrate the presence of neutrophils in the samples tested. Our present results show that the level of MPO in CRS associated with biofilm is lower than that without biofilm. It may suggest either a low number of neutrophils or the presence of a type of neutrophils with compromised antimicrobial activity, described as biofilm-associated neutrophils (BAN). Finally, we conclude that further studies with a large number of CRS cases should be performed to establish the association between S. epidermidis and other frequently isolated bacterial species from paranasal sinuses, with the severity of CRS, biofilm formation and the infiltration of BAN.

Pan_02 murine pancreatic cancer model.

AIM: The aim of this study was to investigate and describe basic features of Pan_02 murine pancreatic ade- nocarcinoma tumor model. Pan_02 has very low sensitivity to chemotherapeutics therefore it is very similar to human pancreatic cancers. MATERIALS AND METHODS: Mice were subcutaneously injected with different number of cells and tumor growth was measured. Tumors were also investigated with ultrasonograph VEVO2100 in doppler mode to detect viable and function- al blood vessels. Collected tumor samples were investigated to asses necrosis and blood vessel permeability. RESULTS: We found substantial differences in tumor growth depending on a number of inoculated tumor cells. Mice injected with 0.5 × 106 cells gave the most consistent pattern of growth. All tumors showed substantial vascularisation but bigger tumors were more likely to develop larger blood vessels instead of a more dense network. CONCLUSIONS: Murine Pan_02 cancer model shares many features with human PDAC cancers and therefore it is a good model to study new drugs. Injection of 0.5 × 106 cells gives consistent results although it requires more time for the tumor to grow. It also allows the immune system to adapt. Owing ta good vascularisation, Pan_02 is a good model to study chemotherapy against pancreatic adenocarcinoma but it may not be the best model for immunotherapy since it does not respond to the immune stimulation (unpublished data).

The class A scavenger receptor SR-A/CD204 and the class B scavenger receptor CD36 regulate immune functions of macrophages differently.

SR-A/CD204 and CD36 are major receptors responsible for oxidized lipoproteins uptake by macrophages in atherosclerotic plaques. Both receptors also share the role as receptors for different pathogens, but studies on their signaling have been hampered by the lack of selective ligands. We report that, upon specific ligation by Ab, SR-A does not induce cytokine production, but mediates inhibition of LPS-stimulated production of IL-6 and IL-12/23p40, enhancement of IL-10 release, and has no effect on TNF-α and RANTES production in murine macrophages. In contrast, anti-CD36 Ab alone stimulated production of all these cytokines, with IL-10 production being exceptionally high. Effects of anti-CD36 Ab, except of IL-10 production, were mediated by CD14 and TLR2, whereas those of SR-A ligation by heterotrimeric Gi/o proteins and by phosphatidylinositol 3-kinases. Surprisingly, we found that LPS uptake by macrophages was mediated in part by CD36 cooperating with CD14, whereas SR-A was not involved in this process. Finely, during in vitro Ag presentation to naïve CD4(+) lymphocytes, pre-incubation of macrophages with anti-CD36 Ab enhanced IFN-γ production in the co-culture, but exerted the opposite effect under conditions enabling IL-10 accumulation. In contrast, anti-SR-A Ab was ineffective alone, but reversed the Th1-polarizing effect of LPS.

Enhanced generation of reactive oxygen intermediates by suppressor T cell-derived exosome-treated macrophages.

Macrophages (Mφ) as efficient phagocytes able to present the antigen and playing an effector role induce and orchestrate the immune response also through the release of soluble factors. Recently described T CD8+ cell-derived suppressive exosomes carrying miRNA-150, that act antigen-specifically, seem to inhibit murine contact sensitivity reaction indirectly by affecting antigen presenting cells, especially Mφ. Present studies investigated the influence of suppressive exosomes on secretory activity of Mφ assessed as their ability to generate reactive oxygen intermediates (ROIs), nitric oxide, cytokines as well as their viability and expression of antigen phagocytosis and presentation markers. Interestingly, in vivo and in vitro treatment of Mφ with assayed hapten-specific exosomes affected only ROIs generation, significantly enhancing their production. Current results suggest that ROIs may participate in antigen-specific tolerance mechanism mediated by suppressive T lymphocyte-derived exosome-influenced Mφ, by inhibition of effector T cell proliferation and induction of T regulatory lymphocytes.

Therapeutic vaccines based on genetically modified Salmonella: a novel strategy in cancer immunotherapy.

In the course of evolution, bacteria from the genus Salmonella adapted to survive and multiply in a vertebrate host. Skillful use of bacterial interactions with the host immune system became the basis for the development of modified Salmonella-based therapeutic vaccines. Bacterial genome can be modified to reduce toxicity and to develop or enhance therapeutic activity. Salmonella-based therapeutic vaccines are an attractive and novel alternative for conventional cancer treatment (chemotherapy, radiotherapy, and passive immunotherapy). Live bacteria have the natural ability to sense external environment and penetrate the target tissue. Appropriate strains of Salmonella, infused into experimental animal tumor model, accumulate selectively in solid tumors and inhibit their growth. Moreover, the bacteria can reach tumor areas that are inaccessible for other, passively diffusing therapeutics, e.g., ischemic areas. Thus, bacteria can produce and locally release a natural or recombinant anticancer agent, which enhances their therapeutic effect. S. typhimurium VNP20009 strain is safe, which has been documented in clinical trials. However, the expected therapeutic benefit has not been observed, presumably due to insufficient tumor colonization by bacteria. To enhance colonization of solid tumors, VNP20009 bacteria have been equipped with the ability to express on the surface an antibody fragment specific for carcinoembryonic antigen present on human tumor cells. Additionally, to potentiate antitumor activity, the genetic material of VNP20009 has been engineered to overproduce an endogenous proapoptotic protein, which targets cancer and immune cells promoting tumor growth.

Immunoregulatory potential of exopolysaccharide from Lactobacillus rhamnosus KL37: effects on the production of inflammatory mediators by mouse macrophages.

The ability to produce exopolysaccharides (EPS) is widespread among lactobacilli including Lactobacillus rhamnosus, the commonly used probiotic bacteria. Exopolysaccharides are a major component of the bacterial biofilm with a well-documented impact on adherence of bacteria to host cells. However, their immunoregulatory properties are unknown. The aim of this study was to examine the immunostimulatory potential of EPS derived from L. rhamnosus KL37. We investigated the effect of EPS on the production of inflammatory mediators by mouse peritoneal macrophages and compared it with the effect of Lipopolysaccharide (LPS). Exopolysaccharides, at concentrations higher than those of LPS, stimulated production of both pro-inflammatory (TNF-α, IL-6, IL-12) and anti-inflammatory (IL-10) cytokines. Interestingly, analysis of the balance of TNF-α/IL-10 production showed a potential pro-inflammatory effect of EPS. Furthermore, our data demonstrate that exposure of macrophages to LPS induced a state of hyporesponsiveness, as indicated by reduced production of TNF-α after restimulation with either LPS or EPS ('cross-tolerance'). By contrast, EPS could make cells tolerant only to subsequent stimulation by the same stimulus. We also examined the relationship between TNF-α production and activation of mitogen-activated protein kinases (MAPKs) by EPS and LPS. Pretreatment of macrophages with specific inhibitors of p38 and ERK MAPKs reduced TNF-α production induced by both stimuli to the same extent. In conclusion, these data demonstrate that EPS can effectively stimulate production of inflammatory mediators by macrophages in vitro. However, to predict whether EPS could be clinically useful as an immunomodulatory agent, further in vivo studies with highly purified EPS are necessary.