Preparation of chemically stable allergen-specific sublingual immunotherapy from Egyptian allergens.

BACKGROUND: The term "allergen extracts" refers to solutions of proteins or glycoproteins extracted from source raw materials. OBJECTIVES: This study was planned to prepare chemically stable sublingual immunotherapy from different allergens in Egypt. METHODS: Allergen extraction from raw materials. The concentrated aqueous extract of each allergen was mixed with an equal volume of glycerol. The protein content of the preparations was determined using the modified Lowry assay method. The prepared allergens were stored for 9 months at 2-4°C. Samples were analyzed periodically (0, 3, 6, and 9 months of intervals) adopting the Lowry Assay method. Levels of specific IgE to Chenopodium album antigens were measured in patients' sera by ELISA. RESULTS: The concentration of all prepared allergens, as indicated by the concentration of the protein content, was found to decrease exponentially with time, implying first-order kinetics of degradation. From the values of the slopes of the log plot for each allergen, the half-life time (t1/2 ) and (t1/4 ) values were calculated. The expiration date was considered as the time after which the allergen loses 25% of its potency. The obtained values of t1/4% vary according to the type of vaccine. The most stable one is that of Chenopodium album pollens (2.4 years) and the least stable is that of house dust Mites (9 months). The immunological characters of Chenopodium album extract were stable for at least 6 months. CONCLUSION: Differences exist among allergen extracts made by multiple manufacturers. So, developments in studies on allergen preparation and characterization in a different locality are necessary.

Inhibition of CL-11 reduces pulmonary inflammation in a mouse model of Klebsiella pneumoniae lung infection.

Infection caused by K. pneumoniae is associated with severe inflammation due to stimulation of the innate immune components including the complement system, which is the main player of the innate immune response. Excessive complement-mediated inflammation may cause severe lung injury. Here we clearly show that K. pneumoniae binds to different lectin pathway carbohydrate recognition molecules and activates the complement cascade via the LP. Administration of anti-CL-11 antibodies 6 h before the infection impairs LP functional activity but it shows no effect on the survival time of mice infected with K. pneumoniae. Similarly, no significant difference in bacterial load in blood and lung tissues was observed between mice that received anti-CL-11 and control group treated with an isotype antibody. Interestingly, treatment of mice with anti-CL-11 prior to infection significantly improved histopathological changes and lung injury score induced by K. pneumoniae. Moreover, administration of anti-CL-11 reduced leukocytes infiltration into lung tissues and decreased the levels of the inflammatory mediators TNF-α, IL-6, and IL-1ß in the infected mice. These findings indicate that inhibition of the LP could secure a significant level of protection against lung injury during the infection caused by K. pneumoniae.

ß-lactam resistance associated with ß-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae.

ß-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating ß-lactam resistance in E. coli and K. pneumoniae clinical isolates collected from Mansoura University hospitals, Egypt. A total of 80 isolates, 45 E. coli and 35 K. pneumoniae isolates, were collected and their antibiotic susceptibility was determined by the Disc diffusion method followed by phenotypic and genotypic detection of extended-spectrum ß-lactamases (ESBLs), AmpC ß-lactamase, carbapenemase enzymes. The outer membrane protein porins of all isolates were analyzed and their genes were examined using gene amplification and sequencing. Also, the resistance to complement-mediated serum killing was estimated. A significant percentage of isolates (93.8%) were multidrug resistance and showed an elevated resistance to ß-lactam antibiotics. The presence of either ESBL or AmpC enzymes was high among isolates (83.75%). Also, 60% of the isolated strains were carbapenemase producers. The most frequently detected gene of ESBL among all tested isolates was blaCTX-M-15 (86.3%) followed by blaTEM-1 (81.3%) and blaSHV-1 (35%) while the Amp-C gene was present in 83.75%. For carbapenemase-producing isolates, blaNDM1 was the most common (60%) followed by blaVIM-1 (35%) and blaOXA-48 (13.8%). Besides, 73.3% and 40% of E. coli and K. pneumoniae isolates respectively were serum resistant. Outer membrane protein analysis showed that 93.3% of E. coli and 95.7% of K. pneumoniae isolates lost their porins or showed modified porins. Furthermore, sequence analysis of tested porin genes in some isolates revealed the presence of frameshift mutations that produced truncated proteins of smaller size. ß-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of ß-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on ß-lactams usage to decrease the emergence of resistant strains.

Effect of Biotitania and Titania Addition on Bioactivity and Antibacterial Properties of Calcium Silicate Cement.

Introduction: Nanoparticles are gaining more interest in dentistry for their antimicrobial, physical as well as other properties. This study aimed to evaluate the effect of adding two types of nanoparticles (NPs) on calcium silicate hydraulic cement's (CSHC) unique bioactivity and antibacterial properties. Methods and Materials: Biotitania/AgCl NPs were synthetized and characterized for its morphology, types of formed functional groups and crystalline AgCl using field emission scanning electron microscope (FE-SEM) equipped with energy-dispersive X-ray spectroscopy (EDS), X-ray diffractometer (XRD), Fourier transformation infrared spectroscopy (FT-IR) and thermo-gravimetric analysis (TGA). The former NPs and commercial titania (TiO2) NPs were added (0.5, 1.5 and 3-weight %) to commercial CSHS powder. A total of 140 disk-shaped specimens (10 mm×1 mm) were prepared (seven material groups per each test in addition to the eighth cell control group) to evaluate cell viability and alkaline phosphatase activity (ALP) after 3 and 12 days, respectively. All were incubated with mesenchymal stem cells. Antibacterial efficacy against Streptococcus mutans (S. mutans) was evaluated through the bacterial growth curve slopes while being in direct contact with the tested material groups for 18 h. One-way analysis of variance (ANOVA) and post hoc Tukey's tests were used to analyze the obtained data. Results: Addition of all NPs percentages had no significant effect (P 0.05) on cell viability in comparison to positive control CSHC. Commercial TiO2 NPs (0.5 weight %) had statistically significant lower values (P≤0.05) for bacterial growth curve slope. However, addition of all NPs percentages had significantly improved (P≤0.05) the ALP activity of CSHC with the most prominent effect to 3-weight% biotitania/AgCl NPs. Conclusion: Based on this in vitro study, addition of biotitania/AgCl NPs up to 3-weight% significantly improved the bioactivity of CSHC without having a significant negative impact on its antibacterial efficacy. Interestingly, the addition of commercial TiO2 even in small amounts can significantly improve CSHC antibacterial efficacy.

Immunization with proline rich region of pneumococcal surface protein A has no role in protection against Streptococcus pneumoniae serotype 19F.

Pneumococcal surface protein A (PspA) is one of the major virulence factors expressed by almost all pneumococcal serotypes and was suggested to be a promising universal vaccine candidate for all pneumococcal sero-groups. Here, we expressed and purified the proline-rich region (PR) of PspA and tested it as a recombinant vaccine against infection caused by a clinical isolate (SP19) of Streptococcus pneumoniae serotype 19F. Our results showed that BALB/c mice immunized with recombinant proline-rich (rPR) region showed a significant higher antibody titre against rPR region compared to control non-immunized group. However, immunized mice or mice recived polyclonal antibodies against rPR region challenged via the intra-peritoneal route with a lethal dose of SP19 isolate showed no significant difference in survival compared to control non-immunized group. These results suggested that, immunization of BALB/c mice with rPR region of PspA is not protective against infection caused by serotype 19F in a mouse model.

Cellular interaction and antibacterial efficacy of two hydraulic calcium silicate-based cements: Cell-dependent model.

BACKGROUND: This study investigated cytotoxic probability, osteogenic potential, and antibacterial efficacy of two pulp-capping hydraulic calcium-silicate cements. MATERIALS AND METHODS: For osteogenic potential and cytotoxicity evaluation, mesenchymal stem cells (MSCs) and materials disc-shaped specimens were used. Increase or decrease in a number of proliferating MSCs was calculated after three intervals. Alkaline phosphatase (ALP) levels in osteogenic media were normalized to the total protein content of cells and measured spectrophotometrically. Antibacterial efficiency through growth curves of Streptococcus mutans in direct contact with tested materials. RESULTS: Biodentine showed the highest number of proliferating MSCs (278000.41 ± 4000.06, after 72 h) and the highest concentration of ALP after 12 days (209.26 ± 7.17 µU/µg protein). It showed the lowest slope (0.003 ± 0.0005) of S. mutans strains growth curves after 18 h. CONCLUSION: Biodentine proved a highly significant osteogenic ability and gave a significant reduction of S. mutans growth.

Bacteriophage Therapy Increases Complement-Mediated Lysis of Bacteria and Enhances Bacterial Clearance After Acute Lung Infection With Multidrug-Resistant Pseudomonas aeruginosa.

Emergence of multidrug-resistant (MDR) bacterial infections is a major problem in clinical medicine. Development of new strategies such as phage therapy may be a novel approach for treatment of life-threatening infections caused by MDR bacteria. A newly isolated phage, MMI-Ps1, with strong lytic activity was used for treatment of acute lung infection with Pseudomonas aeruginosa in a mouse model. Intranasal administration of a single dose of MMI-Ps1 immediately after infection provided a significant level of protection and increased the survival duration. Moreover, treatment of infected mice with phage as late as 12 hours after infection was still protective. Our in vitro results are the first to show the synergistic elimination of serum-resistant Pseudomonas strains by phage and complement. Phage therapy increases the efficacy of complement-mediated lysis of serum-resistant P. aeruginosa strains, indicating the importance of an intact complement system in clearing Pseudomonas infection during phage therapy.

Recombinant chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) protects against LPS-induced lung injury in mice.

Acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) are clinical conditions caused by trauma, lung infection or sepsis. ALI/ARDS is associated with massive recruitment of neutrophils into the lung with release of reactive oxygen species and excessive inflammatory response that damage alveolar tissue. Here we report the successful use of a potent recombinant chemotaxis inhibitory protein (rCHIPS) derived from Staphylococcus aureus in reducing the severity of ALI/ARDS. Treatment with rCHIPS reduces pulmonary inflammation and permeability in mice after intranasal administration of lipopolysaccharide (LPS). rCHIPS treatment significantly reduces lung myeloperoxidase (MPO) activity, pro-inflammatory cytokines, broncho-alveolar lavage (BAL) fluid protein content as well as histopathological changes. In addition, treatment with rCHIPS significantly diminishes neutrophils and leukocytes recruitment into lung tissue after LPS administration and hence protects mice from reactive oxygen species mediated lung injury. Our finding reveals potential therapeutic benefits of using rCHIPS for the treatment of ALI/ARDS.

Immunization with outer membrane proteins (OprF and OprI) and flagellin B protects mice from pulmonary infection with mucoid and nonmucoid Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, which considered as a common cause of nosocomial infection and life-threatening complications in immunocompromized and cystic fibrosis patients. Here, we evaluate the protective effect of recombinant vaccines composed of outer membrane proteins OprF and OprI alone or in combination with flagellin B against mucoid and nonmucoid pseudomonas infection. METHODS: BALB/C mice were immunized subcutaneous using OprF and OprI with or without flagellin B and antibody titers were determined. Serum bactericidal and opsonophagocytosis activities of immunized and control sera were estimated against mucoid and nonmucoid pseudomonas strains. Lung tissue sections from immunized and nonimmunized mice were analyzed and the levels of peripheral neutrophils infiltration into the lung and tissue inflammation were scored. RESULTS: Subcutaneous immunization using OprF and OprI with or without flagellin B elicited higher antibody titers against OprF, OprI, and flagellin B. The produced antibodies successfully opsonized both mucoid and nonmucoid strains with subsequent activation of the terminal pathway of complement that enhances killing of nonmucoid strains via complement-mediated lysis. Furthermore, opsonized mucoid and nonmucoid strains showed enhanced opsonophagocytosis via human peripheral neutrophils, a mechanism that kills P. aeruginosa when complement mediated lysis is not effective especially with mucoid strains. Immunized mice also showed a significant prolonged survival time, lower bacteremia, and reduced lung damage when compared with control nonimmunized mice. CONCLUSION: Our data showed that mice immunized with OprF/OprI or OprF/OprI and flagellin B are significantly protected from infection caused by mucoid and nonmucoid strains of P. aeruginosa.