Comparative study of immune responses elicited by outer membrane vesicles of different Pseudomonas aeruginosa strains.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic mucosal human pathogen that naturally releases outer membrane vesicles (OMVs) in different environments, as do other Gram-negative bacteria. The intestinal tract infections caused by P. aeruginosa increase the risk of respiratory infections and mortality of other diseases related to gut infections. Therefore, in this study, we attempted to investigate toll-like receptor (TLR) signaling pathways and immune response profiles of human colon adenocarcinoma (Caco2) cell line exposed to the OMVs of three different P. aeruginosa strains (i.e., antibiotic-susceptible, multi-drug resistant (MDR), and standard lab ATCC 17933). MATERIALS AND METHODS: Real-time quantitative reverse transcription PCR array was carried out to determine mRNA expression in 84 TLRs signaling pathway genes, and the production of specific cytokines was measured by ELISA. RESULTS: OMVs of different strains could induce unique changes in regulating TLRs signaling pathways, such that there were remarkable differences in pro-inflammatory effects and anti-inflammatory responses among the three strains. CONCLUSION: The more complete immune responses observed through the MAMPs caused by MDR strain OMVs interactions with Caco2 lead us to the conclusion that the use of MDR or cystic fibrosis strain OMVs for better known the host immune responses seems preferable.

Evaluation of 99m Tc-MccJ25 peptide analog in mice bearing B16F10 melanoma tumor as a diagnostic radiotracer.

OBJECTIVES: Despite recent advances in treatment modalities, cancer remains a major source of morbidity and mortality throughout the world. Currently, the development of sensitive and specific molecular imaging probes for early diagnosis of cancer is still a problematic challenge. Previous studies have been shown that some of the antimicrobial peptides (AMPs) exhibit a broad spectrum of cytotoxic activity against cancerous cells in addition to their antimicrobial activities. MicrocinJ25 (MccJ25) is an antimicrobial peptide that is produced by Escherichia coli (E. coli) strain. The aim of this study was to investigate the potential of a new peptide radiopharmaceutical derived from MccJ25 for diagnosis of melanoma tumor bearing C57BL/6 mice. METHODS: A 14 amino acid analog of MccJ25 was labeled with technetium-99m (99mTc) through hydrazinonicotinamide (HYNIC) chelator and tricine as coligand. In vivo tumor uptake and tissue distribution were evaluated. The in vivo biodistribution studies were determined in C57BL/6 mice bearing B16F10 tumor. RESULTS: The amount of non-peptide related 99mTc-impurities that measured by thin layer chromatography (TLC) did not exceed 5% of the total radioactivity. The in vitro binding to B16F10 cells was 30.73 ± 0.9% after 1 h incubation at 37°C, and saturation binding experiments showed good affinity for radio-complex (Kd=47.98±6.25 nM). The melanoma tumor was clearly visible up 1 h post-injection by gamma camera imaging. CONCLUSION: The results showed that 99mTc-labeld peptide could be a promising candidate as a targeting radiopharmaceutical for melanoma tumor imaging in mice.

Synthesis of a Peptide Derivative of MicrocinJ25 and Evaluation of Antibacterial and Biological Activities.

MicrocinJ25 (MccJ25) is a small ribosomally synthesized antimicrobial peptide that is produced by Enterobacteriacea family especially E. coli. The present study focuses on preparation and evaluation of in-vitro antimicrobial and biological properties of a new peptide derived from MccJ25. We prepared a MccJ25-derived peptide containing 14 amino acids and a single intra-molecular disulfide bond according to solid phase synthesis strategy. The purified peptide was characterized by Liquid chromatography-mass spectrometry (LC-MS) and Fourier Transform Infrared (FTIR) spectroscopy. 96-well microdilution plate assay was exerted for determination of minimum inhibitory concentration (MIC) of peptide against different bacterial strains. Cytotoxicity of the peptide derivative on HT-29 cell line assayed using MTT test. The final peptide successfully was prepared with purity more than 99.8% as determined by analytical HPLC. The evaluation of antibacterial activity of the peptide against Gram-positive and Gram- negative bacteria revealed that the peptide was very effective against E. coli 35218 with minimum inhibitory concentration (MIC) at dose 3.9 µM. The hemolytic activity toward human erythrocytes was very minimal below 0.3%. The cell viability percentage of HT-29 cell line after 24 h of contact with the peptide was more than 83%. The high sensitivity of E. coli strain to this new peptide derived from MccJ25 and through minimal toxicity to cancerous cell, suggesting that above synthesized peptide could be considered as a bioactive compound for further investigations.

Three Tests Used to Identify Non-Culturable Form of Helicobacter pylori in Water Samples.

BACKGROUND: Helicobacter pylori, causing the most common chronic bacterial infection, exist in two forms; bacilli and coccoid. The coccoid form is identified as viable but non-culturable bacteria. OBJECTIVES: The current study aimed to conduct culture, polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP) tests to identify coccoid forms of H. pylori. MATERIALS AND METHODS: The PCR and LAMP tests were optimized using specific primers for glmM gene. The sensitivity and specificity of the tests were determined. The current experimental study was conducted on 10 different strains isolated from clinical cases (H1-H10). The isolates were added to tap water and incubated at three different temperatures for one and two months intervals. After pure-culturing of the bacteria, DNAs were extracted and PCR and LAMP were performed. RESULTS: Ten copies of targeted DNA were required for PCR detection whereas only five copies gave a positive reaction by LAMP assay, with 100% specificity. Of the 10 isolates inoculated in water for one and two months at three different temperatures 4, 22, and 37°C, only three cases (5%) were found positive in the first month; 13 (21.6%) and 29 cases (48.3%) were also positive by PCR and LAMP tests in the first and second months. CONCLUSIONS: Results of the current study confirmed that molecular methods such as PCR and LAMP were much more sensitive, rapid, and specific than culturing to identify non-culturable coccoid forms of H. pylori in water.

Expression analysis elucidates the roles of MAML1 and Twist1 in esophageal squamous cell carcinoma aggressiveness and metastasis.

BACKGROUND: Epithelial-mesenchymal transition has recently attracted great attention in studying the malignant progression of cells through a converging pathway of oncogenesis and metastasis. Twist1 and Mastermind-like 1 (MAML1) are major regulators of EMT through different pathways. The aim of this study was to investigate the clinicopathological relevance of the expression of MAML-1 and Twist1 genes in esophageal squamous cell carcinoma (ESCC). METHODS: Tumoral and corresponding normal tissues from 55 treatment-naive ESCC patients were subjected for expression analysis with quantitative real-time RT-PCR. RESULTS: Overexpression of MAML-1 and Twist1 were significantly associated with lymph node metastasis and the surgical staging of tumor. Overexpression of Twist1 was associated with tumor depth of invasion. Mean relative expression (MRE) of MAML1 was significantly higher in patients with metastasis to lymph nodes (3.07 ± 0.51 vs. 0.86 ± 0.58, P = .008). MRE of Twist1 was significantly higher in patients with invasion of tumor to adventitia (T3, T4) (1.97 ± 0.29 vs. 0.39 ± 0.73, P = .036). In advanced stages of tumor (stage III, IV), a significantly higher MRE of Twist1 (2.47 ± 0.41 vs. 1.25 ± 0.36, P = .035) and MAML1 (3.05 ± 0.45 vs. 1.07 ± 0.59, P = .021) mRNA was observed. CONCLUSIONS: We introduce Twist1 and MAML1 as new molecular markers of advanced tumor, which determine the characteristics and aggressive behavior of ESCC. Along with the emerging evidence of their role in different cellular processes and aberrations in various cancers, they are suggested as potentially interesting therapeutic targets to reverse a broad spectrum of functional aberrations that promote ESCC development.

Micromorphology of glandular hairs, biological activity and composition of the essential oil of Tanacetum fisherae (Asteraceae-Anthemideae) from Iran.

The essential oil was obtained from the aerial flowering parts of Tanacetum fisherae, a new record from Iran, by hydrodistillation and analyzed by GC and GC-MS. Eleven components representing 99.9% of the total oil were identified. 1,8-Cineole (79.9%) was characterized as the principal compound. The essential oil and its main component were tested against nine microbial strains. The result of the bioassays revealed that the oil possess potent antimicrobial property. This may be associated due to the presence of 1,8-cineole in the oil which tested individually and its high activity was observed. Micromorphological studies of hairs by scanning electron microscopy (SEM) exhibited the presence of abundant sessile capitate glandular and medifixed T-shaped eglandular trichomes on the leaves, flowers and achene, giving useful diagnostic characters for identification of this medicinal plant.

Identification of cyclophilin as a novel allergen from Platanus orientalis pollens by mass spectrometry.

Oriental plane trees are an important source of airborne allergens in cities of southwest Asia. In spite of extensive studies on Platanus acerifolia allergy, there are no reports on the molecular characterization of pollen allergens from Platanus orientalis trees. In this study, a newly recognized member of cyclophilin family with a molecular weight of 18 kDa was identified as being partly responsible for IgE reactivity of P. orientalis pollen extract.

Oriental plane pollen allergy: identification of allergens and cross-reactivity between relevant species.

Pollen from different tree and grass species represent the greatest inducers of type I allergy worldwide. Oriental plane trees, as Platanus orientalis, are an important source of airborne allergens in cities of the southwest Asia and southeast Europe. This study was aimed to identify relevant allergens of P. orientalis pollen and to ascertain whether P. orientalis allergens have cross-reactivity with related plane trees, such as Platanus acerifolia and Platanus occidentalis pollen components. Nineteen patients with a clinical history of reaction to P. orientalis pollen and a positive skin-prick test (SPT) to P. orientalis pollen extract were included in this study. Identification of IgE-binding proteins in Platanus pollen extracts was elucidated by immunoblotting using sera from P. orientalis pollen-sensitive patients. Cross-reactivity studies among P. orientalis allergens and relevant species was evaluated by immunoblot-inhibition and ELISA inhibition assays. All the patients were polysensitive and exhibited positive SPT to grass and tree pollen allergens. The IgE-binding pattern of P. orientalis pollen extract was well defined. The inhibition experiments showed that the capacity of P. occidentalis was greater than P. acerifolia pollen extract in preventing the reactivity of immobilized P. orientalis pollen extract with the IgE antibodies of patients. The 28-kDa molecule was the most frequent allergen among nine IgE-binding proteins of P. orientalis pollen. The IgE-reactive components of P. orientalis pollen showed a higher level of cross-reactivity with P. occidentalis pollen in comparison with P. acerifolia pollen.