ABSTRACT
BACKGROUND: The resistance of Helicobacter pylori to the antibiotic therapy poses the problem to discover new therapeutic approaches. Recently it has been stated that antibacterial, immunomodulatory, and antioxidant properties of lactoferrin are increased when this protein is surface-linked to biomimetic hydroxyapatite nanocrystals. OBJECTIVE: Based on these knowledge, the aim of the study was to investigate the efficacy of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles with cell free supernatant from probiotic Lactobacillus paracasei as an alternative therapy against Helicobacter pylori infection. METHODS: Antibacterial and antinflammatory properties, humoral antibody induction, histopathological analysis and absence of side effects were evaluated in both in vitro and in vivo studies. RESULTS: The tests carried out have been demonstrated better performance of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles combined with cell free supernatant from probiotic Lactobacillus paracasei compared to both lactoferrin and probiotic alone or pooled. CONCLUSION: These findings indicate the effectiveness and safety of our proposed therapy as alternative treatment for Helicobacter pylori infection.
Subject(s)
Durapatite/chemistry , Helicobacter Infections/microbiology , Helicobacter pylori/physiology , Lactoferrin/chemistry , Nanoparticles/chemistry , Adsorption , Animals , Anti-Infective Agents/administration & dosage , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Biomimetic Materials/chemistry , Culture Media, Conditioned/pharmacology , Cytokines/metabolism , Drug Delivery Systems/methods , Gastric Mucosa/metabolism , Helicobacter Infections/prevention & control , Helicobacter pylori/drug effects , Host-Pathogen Interactions/drug effects , Lacticaseibacillus paracasei/physiology , Lactoferrin/administration & dosage , Lactoferrin/pharmacology , Mice, Inbred BALB C , Microscopy, Electron, Transmission , Nanoparticles/ultrastructure , Probiotics/administration & dosage , Probiotics/pharmacology , Stomach/drug effects , Stomach/microbiology , Treatment Outcome , X-Ray DiffractionABSTRACT
The emergence of bacterial strains resistant to antibiotics is a general public health problem. Progress in developing new molecules with antimicrobial properties has been made. In this study, we evaluated the biological activity of a hybrid nanocomposite composed of synthetic biomimetic hydroxyapatite surface-functionalized by lactoferrin (LF-HA). We evaluated the antimicrobial, anti-inflammatory, and antioxidant properties of LF-HA and found that the composite was active against both Gram-positive and Gram-negative bacteria, and that it modulated proinflammatory and anti-inflammatory responses and enhanced antioxidant properties as compared with LF alone. These results indicate the possibility of using LF-HA as an antimicrobial system and biomimetic hydroxyapatite as a candidate for innovative biomedical applications.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , Lactoferrin/chemistry , Lactoferrin/pharmacology , Nanoparticles/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antioxidants/chemistry , Antioxidants/pharmacology , Cattle , Cell Line, Tumor , Durapatite/chemistry , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Microbial Sensitivity Tests , Nanocomposites/chemistry , Nanomedicine , Nanoparticles/ultrastructureABSTRACT
The study demonstrates that in cattle, animals heterozygous at the MyD88 A625C polymorphic marker have a 5-fold reduced risk for active pulmonary tuberculosis (odds ratio [OR] = 0.19; P = 6 × 10(-12)). The reduced risk, however, does not extend to animals with latent pulmonary tuberculosis (OR = 0.83; P = 0.40). Heterozygosity at the A625C single nucleotide polymorphism is associated with intermediate levels of tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase (NOS). Accordingly, deficiency as well as overexpression of proinflammatory cytokines or NOS favor tuberculosis, while heterozygosity provides the animals with the optimal level of inflammation.
Subject(s)
Genetic Predisposition to Disease , Mycobacterium bovis , Myeloid Differentiation Factor 88/genetics , Polymorphism, Genetic , Tuberculosis, Bovine/genetics , Animals , Base Sequence , Case-Control Studies , Cattle , Female , Genotype , Heterozygote , Inflammation/genetics , Molecular Sequence Data , Tuberculosis, Bovine/immunologyABSTRACT
BACKGROUND: Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs. METHODS: Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR. RESULTS: TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix. CONCLUSION: Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides. GENERAL SIGNIFICANCE: The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.
Subject(s)
Amphibian Proteins , Anti-Bacterial Agents , Drug Design , Escherichia coli/growth & development , Proteins , Amphibian Proteins/chemical synthesis , Amphibian Proteins/chemistry , Amphibian Proteins/pharmacology , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Proteins/chemical synthesis , Proteins/chemistry , Proteins/pharmacology , Rana temporariaABSTRACT
BACKGROUND: Antimicrobial peptides (AMPs) are an ancient group of defense molecules. AMPs are widely distributed in nature (being present in mammals, birds, amphibians, insects, plants, and microorganisms). They display bactericidal as well as immunomodulatory properties. The aim of this study was to investigate the antimicrobial and anti-inflammatory activities of a combination of two AMPs (temporin B and the royal jellein I) against Staphylococcus epidermidis. RESULTS: The temporin B (TB-KK) and the royal jelleins I, II, III chemically modified at the C terminal (RJI-C, RJII-C, RJIII-C), were tested for their activity against 10 different Staphylococcus epidermidis strains, alone and in combination. Of the three royal jelleins, RJI-C showed the highest activity. Moreover, the combination of RJI-C and TB-KK (MIX) displayed synergistic activity. In vitro, the MIX displayed low hemolytic activity, no NO2- production and the ability to curb the synthesis of the pro-inflammatory cytokines TNF-α and IFN-γ to the same extent as acetylsalicylic acid. In vivo, the MIX sterilized mice infected with Staphylococcus epidermidis in eleven days and inhibited the expression of genes encoding the prostaglandin-endoperoxide synthase 2 (COX-2) and CD64, two important parameters of inflammation. CONCLUSION: The study shows that the MIX - a combination of two naturally occurring peptides - displays both antimicrobial and anti-inflammatory activities.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Administration, Intravenous , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Anti-Infective Agents/therapeutic use , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Antimicrobial Cationic Peptides/administration & dosage , Antimicrobial Cationic Peptides/therapeutic use , Bacterial Load/drug effects , Cell Death/drug effects , Disease Models, Animal , Flow Cytometry , Gene Expression Regulation/drug effects , Gentamicins/pharmacology , Hemolysis/drug effects , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Microbial Sensitivity Tests , Molecular Sequence Data , Nitrates/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcal Infections/pathology , Staphylococcus epidermidis/drug effectsABSTRACT
Staphylococcus aureus, in spite of antibiotics, is still a major human pathogen causing a wide range of infections. The present study describes the new vaccine A170PG, a peptidoglycan-based vaccine. In a mouse model of infection, A170PG protects mice against a lethal dose of S. aureus. Protection lasts at least 40 weeks and correlates with increased survival and reduced colonization. Protection extends into drug-resistant (MRSA or VISA) and genetically diverse clinical strains. The vaccine is effective when administered - in a single dose and without adjuvant - by the intramuscular, intravenous or the aerosol routes and induces active as well as passive immunization. Of note, A170PG also displays therapeutic activity, eradicating staphylococci, even when infection is systemic. Sustained antibacterial activity and induction of a strong and rapid anti-inflammatory response are the mechanisms conferring therapeutic efficacy to A170PG.
Subject(s)
Bacterial Vaccines/therapeutic use , Lung Diseases/prevention & control , Peptide Fragments/therapeutic use , Peptidoglycan/therapeutic use , Staphylococcal Infections/prevention & control , Staphylococcus aureus/pathogenicity , Vaccines, Synthetic/therapeutic use , Animals , Antigens, Bacterial/immunology , Chromatography, Affinity , Female , Humans , Lung Diseases/immunology , Lung Diseases/microbiology , Lung Diseases/mortality , Mice , Mice, Inbred BALB C , Peptide Fragments/immunology , Peptidoglycan/immunology , Staphylococcal Infections/immunology , Staphylococcal Infections/microbiology , Staphylococcal Infections/mortality , Staphylococcus aureus/drug effects , Staphylococcus aureus/immunology , Survival Rate , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.
Subject(s)
Bacteriophages/physiology , Staphylococcal Infections/immunology , Staphylococcus aureus/immunology , Staphylococcus aureus/virology , Acetylglucosamine/metabolism , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Vaccines/immunology , Bacteriophages/growth & development , Chromatography, Liquid , Electrophoresis, Gel, Pulsed-Field , Female , Methicillin-Resistant Staphylococcus aureus/immunology , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Mice , Mice, Inbred BALB C , Reverse Transcriptase Polymerase Chain Reaction , Staphylococcal Infections/microbiology , Staphylococcus aureus/pathogenicityABSTRACT
BACKGROUND: The most serious criticisms leveled at bacteriophage therapy are as follows: phages induce neutralizing antibodies, phages are active only when administered shortly after bacterial infection, and phage-resistant bacteria emerge rapidly in the course of therapy. METHODS: Phages lytic for several Salmonella enterica serovars were isolated by means of standard protocols from feces of patients with gastroenteritis. Growth of S. enterica serovar Paratyphi B (Salp572(phi1S)) in the presence of phage phi1 (selected from among 8 phages for its larger host range) provided a phage phi1-resistant bacterial strain (Salp572(phi1R)). The properties of the Salp572(phi1S) and Salp572(phi1R) strains and of phage phi1 were studied in a mouse model of experimental infection. RESULTS: Phages induced nonneutralizing antibodies and were active 2 weeks after experimental infection of mice; phage-resistant bacteria were avirulent and short lived in vivo. More importantly, phage-resistant bacteria were excellent vaccines, protecting against lethal doses of heterologous S. enterica serovars. CONCLUSIONS: Phage therapy effectiveness has not yet been properly assessed.
Subject(s)
Bacteriophages/isolation & purification , Gastroenteritis/microbiology , Gastroenteritis/therapy , Probiotics/therapeutic use , Salmonella paratyphi B , Animals , Antibody Specificity , Bacteriophages/immunology , Disease Models, Animal , Female , Mice , Transformation, BacterialABSTRACT
Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10-14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3-6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.