Presepsin as a biomarker of bacterial translocation and an indicator for the prescription of probiotics in cirrhosis.

BACKGROUND: The gut-liver axis and bacterial translocation are important in cirrhosis, but there is no available universal biomarker of cellular bacterial translocation, for which presepsin may be a candidate. AIM: To evaluate the relationship of the blood presepsin levels with the state of the gut microbiota in cirrhosis in the absence of obvious infection. METHODS: This study included 48 patients with Child-Pugh cirrhosis classes B and C and 15 healthy controls. The fecal microbiome was assessed using 16S rRNA gene sequencing. Plasma levels of presepsin were measured. A total of 22 patients received a probiotic (Saccharomyces boulardii) for 3 months. RESULTS: Presepsin levels were higher in patients with cirrhosis than in healthy individuals [342 (91-2875) vs 120 (102-141) pg/mL; P = 0.048]. Patients with elevated presepsin levels accounted for 56.3% of all included patients. They had lower levels of serum albumin and higher levels of serum total bilirubin and overall severity of cirrhosis as assessed using the Child-Pugh scale. Patients with elevated presepsin levels had an increased abundance of the main taxa responsible for bacterial translocation, namely Bacilli and Proteobacteria (including the main class Gammaproteobacteria and the minor taxa Xanthobacteraceae and Stenotrophomonas), and a low abundance of bacteria from the family Lachnospiraceae (including the minor genus Fusicatenibacter), which produce short-chain fatty acids that have a positive effect on intestinal barrier function. The presepsin level directly correlated with the relative abundance of Bacilli, Proteobacteria, and inversely correlated with the abundance of Lachnospiraceae and Propionibacteriaceae. After 3 months of taking the probiotic, the severity of cirrhosis on the Child-Pugh scale decreased significantly only in the group with elevated presepsin levels [from 9 (8-11) to 7 (6-9); P = 0.004], while there were no significant changes in the group with normal presepsin levels [from 8 (7-8) to 7 (6-8); P = 0.123]. A high level of presepsin before the prescription of the probiotic was an independent predictor of a greater decrease in Child-Pugh scores (P = 0.046), as well as a higher level of the Child-Pugh scale (P = 0.042), but not the C-reactive protein level (P = 0.679) according to multivariate linear regression analysis. CONCLUSION: The level of presepsin directly correlates with the abundance in the gut microbiota of the main taxa that are substrates of bacterial translocation in cirrhosis. This biomarker, in the absence of obvious infection, seems important for assessing the state of the gut-liver axis in cirrhosis and deciding on therapy targeted at the gut microbiota in this disease.

First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

Gut Microbiota and Biomarkers of Endothelial Dysfunction in Cirrhosis.

Our aim was to study the association of endothelial dysfunction biomarkers with cirrhosis manifestations, bacterial translocation, and gut microbiota taxa. The fecal microbiome was assessed using 16S rRNA gene sequencing. Plasma levels of nitrite, big endothelin-1, asymmetric dimethylarginine (ADMA), presepsin, and claudin were measured as biomarkers of endothelial dysfunction, bacterial translocation, and intestinal barrier dysfunction. An echocardiography with simultaneous determination of blood pressure and heart rate was performed to evaluate hemodynamic parameters. Presepsin, claudin 3, nitrite, and ADMA levels were higher in cirrhosis patients than in controls. Elevated nitrite levels were associated with high levels of presepsin and claudin 3, the development of hemodynamic circulation, hypoalbuminemia, grade 2-3 ascites, overt hepatic encephalopathy, high mean pulmonary artery pressure, increased abundance of Proteobacteria and Erysipelatoclostridium, and decreased abundance of Oscillospiraceae, Subdoligranulum, Rikenellaceae, Acidaminococcaceae, Christensenellaceae, and Anaerovoracaceae. Elevated ADMA levels were associated with higher Child-Pugh scores, lower serum sodium levels, hypoalbuminemia, grade 2-3 ascites, milder esophageal varices, overt hepatic encephalopathy, lower mean pulmonary artery pressure, and low abundance of Erysipelotrichia and Erysipelatoclostridiaceae. High big endothelin-1 levels were associated with high levels of presepsin and sodium, low levels of fibrinogen and cholesterol, hypocoagulation, increased Bilophila and Coprobacillus abundances, and decreased Alloprevotella abundance.

Efficacy and Safety of a Probiotic Containing Saccharomyces boulardii CNCM I-745 in the Treatment of Small Intestinal Bacterial Overgrowth in Decompensated Cirrhosis: Randomized, Placebo-Controlled Study.

(1) Background: The aim was to evaluate the effectiveness of the probiotic containing Saccharomyces boulardii in the treatment of small intestinal bacterial overgrowth (SIBO) in patients with decompensated cirrhosis. (2) Methods: This was a blinded, randomized, placebo-controlled study. (3) Results: After 3 months of treatment, SIBO was absent in 80.0% of patients in the probiotic group and in 23.1% of patients in the placebo group (p = 0.002). The patients with eliminated SIBO had decreased frequency of ascites and hepatic encephalopathy, the increased platelets and albumin levels, the decreased blood levels of total bilirubin, biomarkers of bacterial translocation (lipopolysaccharide [LPS]) and systemic inflammation (C-reactive protein), and positive changes in markers of hyperdynamic circulation compared with the state at inclusion. There were no significant changes in the claudin 3 level (the intestinal barrier biomarker) in these patients. No significant changes were observed in the group of patients with persistent SIBO. The serum level of nitrate (endothelial dysfunction biomarker) was lower in patients with eradicated SIBO than in patients with persistent SIBO. One (5.3%) patient with eradicated SIBO and six (42.9%) patients with persistent SIBO died within the first year of follow-up (p = 0.007). (4) Conclusions: SIBO eradication was an independent predictor of a favorable prognosis during the first year of follow-up.

Gut Dysbiosis and Hemodynamic Changes as Links of the Pathogenesis of Complications of Cirrhosis.

The aim was to evaluate the relationship between gut dysbiosis and hemodynamic changes (hyperdynamic circulation) in cirrhosis, and between hemodynamic changes and complications of this disease. This study included 47 patients with cirrhosis. Stool microbiome was assessed using 16S rRNA gene sequencing. Echocardiography with a simultaneous assessment of blood pressure and heart rate was performed to assess systemic hemodynamics. Patients with hyperdynamic circulation had more severe cirrhosis, lower albumin, sodium and prothrombin levels, higher C-reactive protein, aspartate aminotransferase and total bilirubin levels, and higher incidences of portopulmonary hypertension, ascites, overt hepatic encephalopathy, hypoalbuminemia, hypoprothrombinemia, systemic inflammation, and severe hyperbilirubinemia than patients with normodynamic circulation. Patients with hyperdynamic circulation compared with those with normodynamic circulation had increased abundance of Proteobacteria, Enterobacteriaceae, Bacilli, Streptococcaceae, Lactobacillaceae, Fusobacteria, Micrococcaceae, Intestinobacter, Clostridium sensu stricto, Proteus and Rumicoccus, and decreased abundance of Bacteroidetes, Bacteroidaceae, Holdemanella, and Butyrivibrio. The systemic vascular resistance and cardiac output values correlated with the abundance of Proteobacteria, Enterobacteriaceae, Bacilli, Streptococcaceae, Lactobacillaceae, Micrococcaceae, and Fusobacteria. Heart rate and cardiac output value were negatively correlated with the abundance of Bacteroidetes. The mean pulmonary artery pressure value was positively correlated with the abundance of Proteobacteria and Micrococcaceae, and negatively with the abundance of Holdemanella.

Differences in Fecal Short-Chain Fatty Acids between Alcoholic Fatty Liver-Induced Cirrhosis and Non-alcoholic (Metabolic-Associated) Fatty Liver-Induced Cirrhosis.

The objective of this study was to investigate the metabolic activity of the gut microbiota in cirrhosis due to different variants of fatty liver disease (alcoholic vs. non-alcoholic [metabolic-associated] one [AFLD and MAFLD]). The present study included 24 patients with alcoholic liver cirrhosis, 16 patients with MAFLD-related cirrhosis, and 20 healthy controls. The level and spectrum of short-chain fatty acids (SCFAs) were determined via gas-liquid chromatography. All patients with cirrhosis showed a decrease in the total content of SCFAs (p < 0.001) and absolute content of acetate (p < 0.001), propionate (p < 0.001), butyrate (p < 0.001), and isovalerate (p < 0.001). In MAFLD cirrhosis, the metabolic activity of the microbiota was significantly altered compared to patients with alcoholic cirrhosis, as evidenced by a lower total SCFA content (p < 0.001) and absolute content of acetate (p < 0.001), propionate (p < 0.001), and butyrate (p < 0.001); a higher relative content of isovalerate (p < 0.001); and a higher IsoCn/Cn ratio (p < 0.001). Various clinical and laboratory parameters correlate differently with fecal SCFAs and their fractions in cirrhosis due to AFLD and MAFLD. SCFA-producing metabolic activity is reduced more in MAFLD cirrhosis than in alcoholic cirrhosis. According to the etiological factors of cirrhosis, disorders of this metabolic activity may be involved in different pathogenetic pathways.

Influence of Silver Nanoparticles on the Growth of Ascitic and Solid Ehrlich Adenocarcinoma: Focus on Copper Metabolism.

The link between copper metabolism and tumor progression motivated us to use copper chelators for suppression of tumor growth. We assume that silver nanoparticles (AgNPs) can be used for lowering bioavailable copper. Our assumption is based on the ability of Ag(I) ions released by AgNPs in biological media and interfere with Cu(I) transport. Intervention of Ag(I) into copper metabolism leads to the replacement of copper by silver in ceruloplasmin and the decrease in bioavailable copper in the bloodstream. To check this assumption, mice with ascitic or solid Ehrlich adenocarcinoma (EAC) were treated with AgNPs using different protocols. Copper status indexes (copper concentration, ceruloplasmin protein level, and oxidase activity) were monitored to assess copper metabolism. The expression of copper-related genes was determined by real-time PCR in the liver and tumors, and copper and silver levels were measured by FAAS. Intraperitoneal AgNPs treatment beginning on the day of tumor inoculation enhanced mice survival, reduced the proliferation of ascitic EAC cells, and suppressed the activity of HIF1α, TNF-α and VEGFa genes. Topical treatment by the AgNPs, which was started together with the implantation of EAC cells in the thigh, also enhanced mice survival, decreased tumor growth, and repressed genes responsible for neovascularization. The advantages of silver-induced copper deficiency over copper chelators are discussed.

Combined Use of Antimicrobial Peptides with Antiseptics against Multidrug-Resistant Bacteria: Pros and Cons.

Antimicrobial peptides (AMPs) are acknowledged as a promising template for designing new antimicrobials. At the same time, existing toxicity issues and limitations in their pharmacokinetics make topical application one of the less complicated routes to put AMPs-based therapeutics into actual medical practice. Antiseptics are one of the common components for topical treatment potent against antibiotic-resistant pathogens but often with toxicity limitations of their own. Thus, the interaction of AMPs and antiseptics is an interesting topic that is also less explored than combined action of AMPs and antibiotics. Herein, we analyzed antibacterial, antibiofilm, and cytotoxic activity of combinations of both membranolytic and non-membranolytic AMPs with a number of antiseptic agents. Fractional concentration indices were used as a measure of possible effective concentration reduction achievable due to combined application. Cases of both synergistic and antagonistic interaction with certain antiseptics and surfactants were identified, and trends in the occurrence of these types of interaction were discussed. The data may be of use for AMP-based drug development and suggest that the topic requires further attention for successfully integrating AMPs-based products in the context of complex treatment. AMP/antiseptic combinations show promise for creating topical formulations with improved activity, lowered toxicity, and, presumably, decreased chances of inducing bacterial resistance. However, careful assessment is required to avoid AMP neutralization by certain antiseptic classes in either complex drug design or AMP application alongside other therapeutics/care products.

Effect of probiotics on hemodynamic changes and complications associated with cirrhosis: A pilot randomized controlled trial.

BACKGROUND: Bacterial translocation exacerbates the hyperdynamic circulation observed in cirrhosis and contributes to a more severe disease course. Probiotics may reduce bacterial translocation and may therefore be useful to redress the circulatory imbalance. AIM: To investigate the effect of probiotics on hemodynamic parameters, systemic inflammation, and complications of cirrhosis in this randomized placebo-controlled trial. METHODS: This single-blind randomized placebo-controlled study included 40 patients with Child-Pugh class B and C cirrhosis; 24 patients received probiotics (Saccharomyces boulardii) for 3 mo, and 16 patients received a placebo over the same period. Liver function and the systemic hemodynamic status were evaluated pre- and post-intervention. Echocardiography and simultaneous blood pressure and heart rate monitoring were performed to evaluate systemic hemodynamic indicators. Cardiac output and systemic vascular resistance were calculated. RESULTS: Following a 3-mo course of probiotics in comparison to the control group, we observed amelioration of hyperdynamic circulation [a decrease in cardiac output (P = 0.026) and an increase in systemic vascular resistance (P = 0.026)] and systemic inflammation [a decrease in serum C-reactive protein levels (P = 0.044)], with improved liver function [an increase in serum albumin (P = 0.001) and a decrease in the value of Child-Pugh score (P = 0.001)] as well as a reduction in the severity of ascites (P = 0.022), hepatic encephalopathy (P = 0.048), and cholestasis [a decrease in serum alkaline phosphatase (P = 0.016) and serum gamma-glutamyl transpeptidase (P = 0.039) activity] and an increase in platelet counts (P < 0.001) and serum sodium level (P = 0.048). CONCLUSION: Probiotic administration was associated with amelioration of hyperdynamic circulation and the associated complications of cirrhosis.

Efficacy and safety of COVID-19 vaccination in patients with cirrhosis.

BACKGROUND: The clinical efficacy and safety of vaccination against novel coronavirus disease 2019 (COVID-19) in patients with cirrhosis have not been evaluated yet. AIM: To evaluate the clinical efficacy and safety of vaccination against COVID-19 in patients with cirrhosis. METHODS: This was a retrospective cohort study of patients with cirrhosis. The first cohort included patients vaccinated with Gam-COVID-Vac (Sputnik V); the second one consisted of unvaccinated controls. RESULTS: The study included 89 vaccinated patients and 148 unvaccinated ones. There were 4 cases of COVID-19 in the vaccinated group and 24 cases in the unvaccinated group (P = 0.035). No severe cases of COVID-19 were revealed in the vaccinated group, while there were 12 ones in the unvaccinated group (P = 0.012) with 10 deaths detected (P = 0.012). The vaccine efficacy was 69.5% (95% confidence interval [CI]: 18.5%-94.4%) against symptomatic cases of COVID-19, 100% (95%CI: 25.1%-100.0%) against severe cases, and 100% (95%CI: 1.6%-100.0%) against death associated with COVID-19. The efficacy of full vaccination with revaccination against symptomatic cases of COVID-19 was 88.3% (95%CI: 48.0%-99.6%). The overall mortality rate was higher in the unvaccinated group than in the vaccinated group (17.1% vs 3.0%; P = 0.001). Higher Child-Turcotte-Pugh class cirrhosis (hazard ratio [HR] = 4.13, 95%CI: 1.82-9.35) and higher age (HR = 1.08, 95%CI: 1.04-1.15) were independent predictors of overall mortality, while vaccination had a protective effect (HR = 0.09, 95%CI: 0.01-0.76). There was no significant difference in liver-related mortality (P = 0.135) or the incidence of liver decompensation (P = 0.077), bleeding esophageal varices (P = 0.397), and vascular events (P = 0.651) between the two groups of patients. CONCLUSION: Vaccination against COVID-19 in patients with cirrhosis is effective and safe.

Gut dysbiosis and body composition in cirrhosis.

BACKGROUND: Gut dysbiosis and changes in body composition (i.e., a decrease in the proportion of muscle mass and an increase in extracellular fluid) are common in cirrhosis. AIM: To study the relationship between the gut microbiota and body composition in cirrhosis. METHODS: This observational study included 46 patients with cirrhosis. Stool microbiome was assessed using 16S rRNA gene sequencing. Multifrequency bioelectrical impedance analysis was performed to assess body composition in these patients. RESULTS: An increase in fat mass and a decrease in body cell mass were noted in 23/46 (50.0%) and 15/46 (32.6%) patients, respectively. Changes in the gut microbiome were not independently associated with the fat mass percentage in cirrhosis. The abundance of Bacteroidaceae (P = 0.041) and Eggerthella (P = 0.001) increased, whereas that of Erysipelatoclostridiaceae (P = 0.006), Catenibacterium (P = 0.021), Coprococcus (P = 0.033), Desulfovibrio (P = 0.043), Intestinimonas (P = 0.028), and Senegalimassilia (P = 0.015) decreased in the gut microbiome of patients with body cell mass deficiency. The amount of extracellular fluid increased in 22/46 (47.6%) patients. Proteobacteria abundance (P < 0.001) increased, whereas Firmicutes (P = 0.023), Actinobacteria (P = 0.026), Bacilli (P = 0.008), Anaerovoraceceae (P = 0.027), Christensenellaceae (P = 0.038), Eggerthellaceae (P = 0.047), Erysipelatoclostridiaceae (P = 0.015), Erysipelotrichaceae (P = 0.003), Oscillospiraceae (P = 0.024), Rikenellaceae (P = 0.002), Collinsella (P = 0.030), Hungatella (P = 0.040), Peptococcaceae (P = 0.023), Slackia (P = 0.008), and Senegalimassilia (P = 0.024) abundance decreased in these patients. Patients with clinically significant ascites (n = 9) had a higher abundance of Proteobacteria (P = 0.031) and a lower abundance of Actinobacteria (P = 0.019) and Bacteroidetes (P = 0.046) than patients without clinically significant ascites (n = 37). CONCLUSION: Changes in the amount of body cell mass and extracellular fluid are associated with changes in the gut microbiome in cirrhosis patients.

The Role of Bile Acids in the Human Body and in the Development of Diseases.

Bile acids are specific and quantitatively important organic components of bile, which are synthesized by hepatocytes from cholesterol and are involved in the osmotic process that ensures the outflow of bile. Bile acids include many varieties of amphipathic acid steroids. These are molecules that play a major role in the digestion of fats and the intestinal absorption of hydrophobic compounds and are also involved in the regulation of many functions of the liver, cholangiocytes, and extrahepatic tissues, acting essentially as hormones. The biological effects are realized through variable membrane or nuclear receptors. Hepatic synthesis, intestinal modifications, intestinal peristalsis and permeability, and receptor activity can affect the quantitative and qualitative bile acids composition significantly leading to extrahepatic pathologies. The complexity of bile acids receptors and the effects of cross-activations makes interpretation of the results of the studies rather difficult. In spite, this is a very perspective direction for pharmacology.

A Recent Ten-Year Perspective: Bile Acid Metabolism and Signaling.

Bile acids are important physiological agents required for the absorption, distribution, metabolism, and excretion of nutrients. In addition, bile acids act as sensors of intestinal contents, which are determined by the change in the spectrum of bile acids during microbial transformation, as well as by gradual intestinal absorption. Entering the liver through the portal vein, bile acids regulate the activity of nuclear receptors, modify metabolic processes and the rate of formation of new bile acids from cholesterol, and also, in all likelihood, can significantly affect the detoxification of xenobiotics. Bile acids not absorbed by the liver can interact with a variety of cellular recipes in extrahepatic tissues. This provides review information on the synthesis of bile acids in various parts of the digestive tract, its regulation, and the physiological role of bile acids. Moreover, the present study describes the involvement of bile acids in micelle formation, the mechanism of intestinal absorption, and the influence of the intestinal microbiota on this process.

Early viral versus late antibiotic-associated diarrhea in novel coronavirus infection.

ABSTRACT: Diarrhea is one of the manifestations of the novel coronavirus disease (COVID-19), but it also develops as a complication of massive antibiotic therapy in this disease. This study aimed to compare these types of diarrhea.We included patients with COVID-19 in a cohort study and excluded patients with chronic diarrhea, laxative use, and those who died during the first day of hospitalization.There were 89 (9.3%), 161 (16.7%), and 731 (75.7%) patients with early viral, late antibiotic-associated, and without diarrhea, respectively. Late diarrhea lasted longer (6 [4-10] vs 5 [3-7] days, P < .001) and was more severe. Clostridioides difficile was found in 70.5% of tested patients with late diarrhea and in none with early diarrhea. Presence of late diarrhea was associated with an increased risk of death after 20 days of disease (P = .009; hazard ratio = 4.7). Patients with late diarrhea had a longer hospital stay and total disease duration, and a higher proportion of these patients required intensive care unit admission. Oral amoxicillin/clavulanate (odds ratio [OR] = 2.23), oral clarithromycin (OR = 3.79), and glucocorticoids (OR = 4.41) use was a risk factor for the development of late diarrhea, while ceftriaxone use (OR = 0.35) had a protective effect. Before the development of late diarrhea, decrease in C-reactive protein levels and increase in lymphocyte count stopped but the white blood cell and neutrophil count increased. An increase in neutrophils by >0.6 × 109 cells/L predicted the development of late diarrhea in the coming days (sensitivity 82.0%, specificity 70.8%, area under the curve = 0.791 [0.710-0.872]).Diarrhea in COVID-19 is heterogeneous, and different types of diarrhea require different management.

Silver Nanoparticles Functionalized With Antimicrobial Polypeptides: Benefits and Possible Pitfalls of a Novel Anti-infective Tool.

Silver nanoparticles (AgNPs) and antimicrobial peptides or proteins (AMPs/APs) are both considered as promising platforms for the development of novel therapeutic agents effective against the growing number of drug-resistant pathogens. The observed synergy of their antibacterial activity suggested the prospect of introducing antimicrobial peptides or small antimicrobial proteins into the gelatinized coating of AgNPs. Conjugates with protegrin-1, indolicidin, protamine, histones, and lysozyme were comparatively tested for their antibacterial properties and compared with unconjugated nanoparticles and antimicrobial polypeptides alone. Their toxic effects were similarly tested against both normal eukaryotic cells (human erythrocytes, peripheral blood mononuclear cells, neutrophils, and dermal fibroblasts) and tumor cells (human erythromyeloid leukemia K562 and human histiocytic lymphoma U937 cell lines). The AMPs/APs retained their ability to enhance the antibacterial activity of AgNPs against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, when conjugated to the AgNP surface. The small, membranolytic protegrin-1 was the most efficient, suggesting that a short, rigid structure is not a limiting factor despite the constraints imposed by binding to the nanoparticle. Some of the conjugated AMPs/APs clearly affected the ability of nanoparticle to permeabilize the outer membrane of Escherichia coli, but none of the conjugated AgNPs acquired the capacity to permeabilize its cytoplasmic membrane, regardless of the membranolytic potency of the bound polypeptide. Low hemolytic activity was also found for all AgNP-AMP/AP conjugates, regardless of the hemolytic activity of the free polypeptides, making conjugation a promising strategy not only to enhance their antimicrobial potential but also to effectively reduce the toxicity of membranolytic AMPs. The observation that metabolic processes and O2 consumption in bacteria were efficiently inhibited by all forms of AgNPs is the most likely explanation for their rapid and bactericidal action. AMP-dependent properties in the activity pattern of various conjugates toward eukaryotic cells suggest that immunomodulatory, wound-healing, and other effects of the polypeptides are at least partially transferred to the nanoparticles, so that functionalization of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.

Caprine Bactenecins as Promising Tools for Developing New Antimicrobial and Antitumor Drugs.

Proline-rich antimicrobial peptides (PR-AMPs) having a potent antimicrobial activity predominantly toward Gram-negative bacteria and negligible toxicity toward host cells, are attracting attention as new templates for developing antibiotic drugs. We have previously isolated and characterized several bactenecins that are promising in this respect, from the leukocytes of the domestic goat Capra hircus: ChBac5, miniChBac7.5N-α, and -ß, as well as ChBac3.4. Unlike the others, ChBac3.4 shows a somewhat unusual pattern of activities for a mammalian PR-AMP: it is more active against bacterial membranes as well as tumor and, to the lesser extent, normal cells. Here we describe a SAR study of ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL-NH2) which elucidates its peculiarities and evaluates its potential as a lead for antimicrobial or anticancer drugs based on this peptide. A set of designed structural analogues of ChBac3.4 was explored for antibacterial activity toward drug-resistant clinical isolates and antitumor properties. The N-terminal region was found to be important for the antimicrobial action, but not responsible for the toxicity toward mammalian cells. A shortened variant with the best selectivity index toward bacteria demonstrated a pronounced synergy in combination with antibiotics against Gram-negative strains, albeit with a somewhat reduced ability to inhibit biofilm formation compared to native peptide. C-terminal amidation was examined for some analogues, which did not affect antimicrobial activity, but somewhat altered the cytotoxicity toward host cells. Interestingly, non-amidated peptides showed a slight delay in their impact on bacterial membrane integrity. Peptides with enhanced hydrophobicity showed increased toxicity, but in most cases their selectivity toward tumor cells also improved. While most analogues lacked hemolytic properties, a ChBac3.4 variant with two additional tryptophan residues demonstrated an appreciable activity toward human erythrocytes. The variant demonstrating the best tumor/nontumor cell selectivity was found to more actively initiate apoptosis in target cells, though its action was slower than that of the native ChBac3.4. Its antitumor effectiveness was successfully verified in vivo in a murine Ehrlich ascites carcinoma model. The obtained results demonstrate the potential of structural modification to manage caprine bactenecins' selectivity and activity spectrum and confirm that they are promising prototypes for antimicrobial and anticancer drugs design.

Human antimicrobial peptides in autoimmunity.

Antimicrobial peptides (AMPs) were firstly discovered as cytotoxic substances that killed bacteria. Later they were described as biologically active peptides that are able not only to kill invaders but also to modulate host immunity. In particular, it is shown that human antimicrobial peptides are able to influence the activity of different innate and adaptive immunity components, thus, obviously, they also participate in autoimmune processes. In this review we discuss the nature of human AMPs and analyze their role in such autoimmune disorders like type 1 diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, Crohn's disease and sarcoidosis. These peptides were shown to have a "double-sided" influence on the autoimmune disease pathogenesis. Thus, described facts should be taken into account for the development of new pharmaceutical agents to cure patients with autoimmune disorders. These agents could derive from natural antimicrobial peptides that in some cases modulate immune response. For example, it was shown that human AMPs are able to modulate complement system dysregulation of which is known to be one of the most dangerous pathogenic factors during autoimmune processes.

Redesigning Arenicin-1, an Antimicrobial Peptide from the Marine Polychaeta Arenicola marina, by Strand Rearrangement or Branching, Substitution of Specific Residues, and Backbone Linearization or Cyclization.

Arenicin-1, a ß-sheet antimicrobial peptide isolated from the marine polychaeta Arenicola marina coelomocytes, has a potent, broad-spectrum microbicidal activity and also shows significant toxicity towards mammalian cells. Several variants were rationally designed to elucidate the role of structural features such as cyclization, a certain symmetry of the residue arrangement, or the presence of specific residues in the sequence, in its membranolytic activity and the consequent effect on microbicidal efficacy and toxicity. The effect of variations on the structure was probed using molecular dynamics simulations, which indicated a significant stability of the ß-hairpin scaffold and showed that modifying residue symmetry and ß-strand arrangement affected both the twist and the kink present in the native structure. In vitro assays against a panel of Gram-negative and Gram-positive bacteria, including drug-resistant clinical isolates, showed that inversion of the residue arrangement improved the activity against Gram-negative strains but decreased it towards Gram-positive ones. Variants with increased symmetry were somewhat less active, whereas both backbone-cyclized and linear versions of the peptides, as well as variants with R→K and W→F replacement, showed antimicrobial activity comparable with that of the native peptide. All these variants permeabilized both the outer and the inner membranes of Escherichia coli, suggesting that a membranolytic mechanism of action was maintained. Our results indicate that the arenicin scaffold can support a considerable degree of variation while maintaining useful biological properties and can thus serve as a template for the elaboration of novel anti-infective agents.

Application of Antimicrobial Peptides of the Innate Immune System in Combination With Conventional Antibiotics-A Novel Way to Combat Antibiotic Resistance?

Rapidly growing resistance of pathogenic bacteria to conventional antibiotics leads to inefficiency of traditional approaches of countering infections and determines the urgent need for a search of fundamentally new anti-infective drugs. Antimicrobial peptides (AMPs) of the innate immune system are promising candidates for a role of such novel antibiotics. However, some cytotoxicity of AMPs toward host cells limits their active implementation in medicine and forces attempts to design numerous structural analogs of the peptides with optimized properties. An alternative route for the successful AMPs introduction may be their usage in combination with conventional antibiotics. Synergistic antibacterial effects have been reported for a number of such combinations, however, the molecular mechanisms of the synergy remain poorly understood and little is known whether AMPs cytotoxicy for the host cells increases upon their application with antibiotics. Our study is directed to examination of a combined action of natural AMPs with different structure and mode of action (porcine protegrin 1, caprine bactenecin ChBac3.4, human alpha- and beta-defensins (HNP-1, HNP-4, hBD-2, hBD-3), human cathelicidin LL-37), and egg white lysozyme with varied antibiotic agents (gentamicin, ofloxacin, oxacillin, rifampicin, polymyxin B, silver nanoparticles) toward selected bacteria, including drug-sensitive and drug-resistant strains, as well as toward some mammalian cells (human erythrocytes, PBMC, neutrophils, murine peritoneal macrophages and Ehrlich ascites carcinoma cells). Using "checkerboard titrations" for fractional inhibitory concentration indexes evaluation, it was found that synergy in antibacterial action mainly occurs between highly membrane-active AMPs (e.g., protegrin 1, hBD-3) and antibiotics with intracellular targets (e.g., gentamicin, rifampcin), suggesting bioavailability increase as the main model of such interaction. In some combinations modulation of dynamics of AMP-bacterial membrane interaction in presence of the antibiotic was also shown. Cytotoxic effects of the same combinations toward normal eukaryotic cells were rarely synergistic. The obtained data approve that combined application of antimicrobial peptides with antibiotics or other antimicrobials is a promising strategy for further development of new approach for combating antibiotic-resistant bacteria by usage of AMP-based therapeutics. Revealing the conventional antibiotics that increase the activity of human endogenous AMPs against particular pathogens is also important for cure strategies elaboration.

Fatty Liver and Systemic Atherosclerosis in a Young, Lean Patient: Rule Out Lysosomal Acid Lipase Deficiency.

Lysosomal acid lipase deficiency (LALD) is a rare genetic disease characterized by the accumulation of cholesteryl esters and triglycerides in many organs, including the liver, spleen, lymph nodes, bone marrow, and vascular endothelium. Patients with LALD can appear asymptomatic until liver failure or premature sudden death from coronary artery disease, stroke, and aneurysm, which lead to the diagnosis. Herein, we present a diagnostic workup in a young 17-year-old female patient who manifested hepatosplenomegaly, elevated liver enzymes, severe dyslipidemia, and systemic atherosclerosis. Liver biopsy demonstrated over 90% diffuse microvesicular steatosis, lipid accumulation in Kupffer cells, and birefringent cholesteryl ester crystals. The diagnosis of LALD was proven by the decrease of lysosomal acid lipase activity in dried blood spots and by the detection of two compound heterozygous mutations in the LIPA gene: nonsense mutation G796T (Gly266Term) and splicing site mutation G894A (E8SJM). The patient started enzyme replacement therapy with sebelipase alfa. Following the 1-year treatment, the patient remained asymptomatic, her serum aminotransferase levels were normal, liver density increased due to lipid resorption, and plaque-associated stenosis of carotid artery regressed. Moreover, liver biopsy showed a decrease of cholesteryl ester crystals in Kupffer cells.