Andrzej Namiot

Intestinal-type and liver-type fatty acid-binding protein in the intestine. Tissue distribution and clinical utility.

OBJECTIVES Intestinal-type fatty acid-binding protein (I-FABP) has been proposed as plasma marker for the detection of acute intestinal injury. However, intestinal mucosa also expresses liver-type FABP (L-FABP). We have investigated the tissue distribution of I-FABP and L-FABP in segments of the human intestine along the duodenal to colonal axis and the potential of both proteins to serve as plasma marker for the diagnosis of intestinal injury. DESIGN AND METHODS I-FABP and L-FABP were measured with specific immunoassays in autopsy samples of the intestine (duodenum, jejunum, ileum and colon) of 23 subjects and in plasma samples from patients (n = 51) with intestinal and/or hepatic disease. Plasma reference values were established in normal healthy individuals (n = 92). RESULTS The I-FABP tissue contents in duodenum, jejunum, ileum, proximal colon and distal colon amounted to 2.22, 4.79, 1.04, 0.27 and 0.25 mug/g ww, respectively. L-FABP tissue contents were markedly higher, amounting to 124 and 198 mug/g ww in duodenum and jejunum, and to 58, 26 and 44 mug/g ww in ileum, proximal colon and distal colon, respectively. Elevated plasma levels of both I-FABP and L-FABP were found in patients suffering from intestinal diseases, while only L-FABP was increased in cases of purely hepatocellular injury. CONCLUSIONS I-FABP and L-FABP show a similar pattern of tissue distribution along the duodenal to colonal axis with highest tissue contents found in the jejunum but in each intestinal segment a >40-fold higher content of L-FABP than of I-FABP. Accordingly, besides I-FABP, also L-FABP is a useful plasma marker for the detection of intestinal injury, especially in patients undergoing intestinal surgery.

Cathelicidin LL-37: A Multitask Antimicrobial Peptide

The antimicrobial peptide LL-37 is the only known member of the cathelicidin family of peptides expressed in humans. LL-37 is a multifunctional host defense molecule essential for normal immune responses to infection and tissue injury. LL-37 peptide is a potent killer of different microorganisms with the ability to prevent immunostimulatory effects of bacterial wall molecules such as lipopolysaccharide and can therefore protect against lethal endotoxemia. Additional reported activities of LL-37 include chemoattractant function, inhibition of neutrophil apoptosis, and stimulation of angiogenesis, tissue regeneration, and cytokine release (e.g. IL-8). Cellular production of LL-37 is affected by multiple factors, including bacterial products, host cytokines, availability of oxygen, and sun exposure through the activation of CAP-18 gene expression by vitamin D3. At infection sites, the function of LL-37 can be inhibited by charge-driven interactions with DNA and F-actin released from dead neutrophils and other cells lysed as the result of inflammation. A better understanding of LL-37’s biological properties is necessary for its possible therapeutic application for immunomodulatory purposes as well as in treating bacterial infection.

Potential of ceragenin CSA‐13 and its mixture with pluronic F‐127 as treatment of topical bacterial infections

Aims:  Ceragenin CSA‐13 is a synthetic mimic of cationic antibacterial peptides, with facial amphiphilic morphology reproduced using a cholic acid scaffold. Previous data have shown that this molecule displays broad‐spectrum antibacterial activity, which decreases in the presence of blood plasma. However, at higher concentrations, CSA‐13 can cause lysis of erythrocytes. This study was designed to assess in vitro antibacterial and haemolytic activity of CSA‐13 in the presence of pluronic F‐127.

Bactericidal activities of the cationic steroid CSA-13 and the cathelicidin peptide LL-37 against Helicobacter pylori in simulated gastric juice

BackgroundThe worldwide appearance of drug-resistant strains of H. pylori motivates a search for new agents with therapeutic potential against this family of bacteria that colonizes the stomach, and is associated with adenocarcinoma development. This study was designed to assess in vitro the anti-H. pylori potential of cathelicidin LL-37 peptide, which is naturally present in gastric juice, its optimized synthetic analog WLBU2, and the non-peptide antibacterial agent ceragenin CSA-13.ResultsIn agreement with previous studies, increased expression of hCAP-18/LL-37 was observed in gastric mucosa obtained from H. pylori infected subjects. MBC (minimum bactericidal concentration) values determined in nutrient-containing media range from 100-800 μg/ml for LL-37, 17.8-142 μg/ml for WLBU2 and 0.275-8.9 μg/ml for ceragenin CSA-13. These data indicate substantial, but widely differing antibacterial activities against clinical isolates of H. pylori. After incubation in simulated gastric juice (low pH with presence of pepsin) CSA-13, but not LL-37 or WLBU2, retained antibacterial activity. Compared to LL-37 and WLBU2 peptides, CSA-13 activity was also more resistant to inhibition by isolated host gastric mucins.ConclusionThese data indicate that cholic acid-based antimicrobial agents such as CSA-13 resist proteolytic degradation and inhibition by mucin and have potential for treatment of H. pylori infections, including those caused by the clarithromycin and/or metronidazole-resistant strains.

Cathelicidin LL-37 Increases Lung Epithelial Cell Stiffness, Decreases Transepithelial Permeability, and Prevents Epithelial Invasion by Pseudomonas aeruginosa

In addition to its antibacterial activity, the cathelicidin-derived LL-37 peptide induces multiple immunomodulatory effects on host cells. Atomic force microscopy, F-actin staining with phalloidin, passage of FITC-conjugated dextran through a monolayer of lung epithelial cells, and assessment of bacterial outgrowth from cells subjected to Pseudomonas aeruginosa infection were used to determine LL-37’s effect on epithelial cell mechanical properties, permeability, and bacteria uptake. A concentration-dependent increase in stiffness and F-actin content in the cortical region of A549 cells and primary human lung epithelial cells was observed after treatment with LL-37 (0.5–5 μM), sphingosine 1-phosphate (1 μM), or LPS (1 μg/ml) or infection with PAO1 bacteria. Other cationic peptides, such as RK-31, KR-20, or WLBU2, and the antibacterial cationic steroid CSA-13 did not reproduce the effect of LL-37. A549 cell pretreatment with WRW4, an antagonist of the transmembrane formyl peptide receptor-like 1 protein attenuated LL-37’s ability to increase cell stiffness. The LL-37–mediated increase in cell stiffness was accompanied by a decrease in permeability and P. aeruginosa uptake by a confluent monolayer of polarized normal human bronchial epithelial cells. These results suggested that the antibacterial effect of LL-37 involves an LL-37–dependent increase in cell stiffness that prevents epithelial invasion by bacteria.

Combined Antibacterial and Anti-Inflammatory Activity of a Cationic Disubstituted Dexamethasone-Spermine Conjugate

ABSTRACT The rising number of antibiotic-resistant bacterial strains represents an emerging health problem that has motivated efforts to develop new antibacterial agents. Endogenous cationic antibacterial peptides (CAPs) that are produced in tissues exposed to the external environment are one model for the design of novel antibacterial compounds. Here, we report evidence that disubstituted dexamethasone-spermine (D2S), a cationic corticosteroid derivative initially identified as a by-product of synthesis of dexamethasone-spermine (DS) for the purpose of improving cellular gene delivery, functions as an antibacterial peptide-mimicking molecule. This moiety exhibits bacterial killing activity against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa present in cystic fibrosis (CF) sputa, and Pseudomonas aeruginosa biofilm. Although compromised in the presence of plasma, D2S antibacterial activity resists the proteolytic activity of pepsin and is maintained in ascites, cerebrospinal fluid, saliva, and bronchoalveolar lavage (BAL) fluid. D2S also enhances S. aureus susceptibility to antibiotics, such as amoxicillin (AMC), tetracycline (T), and amikacin (AN). Inhibition of interleukin-6 (IL-6) and IL-8 release from lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated neutrophils in the presence of D2S suggests that this molecule might also prevent systemic inflammation caused by bacterial wall products. D2S-mediated translocation of green fluorescent protein (GFP)-labeled glucocorticoid receptor (GR) in bovine aorta endothelial cells (BAECs) suggests that some of its anti-inflammatory activities involve engagement of glucocorticoid receptors. The combined antibacterial and anti-inflammatory activities of D2S suggest its potential as an alternative to natural CAPs in the prevention and treatment of some bacterial infections.

Patient factors affecting culture of Helicobacter pylori isolated from gastric mucosal specimens

PURPOSE Culture is one of the methods used for detecting Helicobacter pylori in the stomach. However, since it is costly, labor-consuming, and in a number of infected subjects gives a false negative result, the procedure is not routinely used. The aim of the study was to analyze some of the factors that may affect the outcome of H. pylori culture from endoscopic gastric mucosal specimens. MATERIAL AND METHODS The study was conducted in a group of 265 subjects. The culture of gastric mucosal specimens was verified by urease test and histological examination. If the culture result was not consistent with one or two verifying tests, an additional two tests were used, i.e. H. pylori antigens in stool samples and anti-H. pylori antibodies in blood serum. RESULTS In patients infected with H. pylori (at least two positive diagnostic tests), the analysis of factors that may affect the culture outcome revealed that neither age, gender, smoking, history of eradication, endoscopic diagnosis, use of proton pump inhibitors, ultrasonography of the abdomen or chest radiology performed the day before or on the day of gastroscopy, nor preparation for colonoscopy using osmotic fluids 1-2 days prior to gastroscopy had an effect on the culture outcome. Only high activity of gastritis (neutrophil infiltration) and low bacterial load in gastric mucosal specimens as well as drinking alcohol and the use of histamine H₂ receptor blockers reduced culture efficacy in infected subjects. CONCLUSIONS High activity of gastritis, low bacterial load, drinking alcohol and the use of histamine H₂ receptor blockers can be the cause of failed H. pylori culture from gastric mucosa in the infected subjects. These factors should be taken into consideration when qualifying patients for the test and interpreting the results.

Modulation of exogenous antibiotic activity by host cathelicidin LL-37

Leszczyńska K, Namiot A, Janmey PA, Bucki R. Modulation of exogenous antibiotic activity by host cathelicidin LL‐37. APMIS 2010; 118: 830–6.

The use of immunological method for identification of helicobacter pylori in culture

The aim of the study was to establish whether the immune method detecting Helicobacter pylori antigens can be used for the identification of H. pylori from a culture. Bacteria were cultured from endoscopic specimens of gastric mucosa from 378 patients. A positive result of H. pylori culture was obtained in 166 patients (43.9%), while the presence of H. pylori antigens was obtained in 164 (98.8%). The classical method for H. pylori identification from a culture is characterized by high accuracy. Therefore, a positive result of the test for the presence of H. pylori antigens from the culture could only be additional evidence confirming the culture results in selected cases.