Polyclonal anti-whole cell IgY passive immunotherapy shields against P. aeruginosa-induced acute pneumonia and burn wound infections in murine models.

Pseudomonas aeruginosa (PA) is a multidrug-resistant (MDR) opportunistic pathogen causing severe hospital-, and community-acquired infections worldwide. Thus, the development of effective immunotherapy-based treatments is essential to combat the MDR-PA infections. In the current study, we evaluated the protective efficacy of polyclonal avian antibodies raised against inactivated whole cells of the PAO1 strain in murine models of acute pneumonia and burn wound. The efficacy of generated antibodies was evaluated against different PA strains through several in vitro, ex vivo and in vivo experiments. The results showed that the anti-PAO1-IgY effectively reduced the motility, biofilm formation and cell internalization ability, and enhanced the opsonophagocytic killing of PA strains through the formation of immobilized bacteria and induction of increased cell surface hydrophobicity. Furthermore, immunotherapy with anti-PAO1-IgY completely protected mice against all PA strains in both acute pneumonia and burn wound murine models. It was found to reduce the bacterial loads in infected burned mice through interfering with virulence factors that play vital roles in the early stages of PA infection, such as colonization and cell internalization. The immunotherapy with anti-PAO1-IgYs could be instrumental in developing effective therapies aimed at reducing the morbidity and mortality associated with PA infections.

Immunotherapy with IgY Antibodies toward Outer Membrane Protein F Protects Burned Mice against Pseudomonas aeruginosa Infection.

Burn patients with multidrug-resistant Pseudomonas aeruginosa infections commonly suffer from high morbidity and mortality, which present a major challenge to healthcare systems throughout the world. Outer membrane protein F (OprF), as a main outer membrane porin, is required for full virulence expression of P. aeruginosa. The aim of this study was to evaluate the protective efficacy of egg yolk-specific antibody (IgY) raised against recombinant OprF (r-OprF) protein in a murine burn model of infection. The hens were immunized with r-OprF, and anti-r-OprF IgY was purified using salt precipitation. Groups of mice were injected with different regimens of anti-OprF IgY or control IgY (C-IgY). Infections were caused by subcutaneous injection of P. aeruginosa strain PAO1 at the burn site. Mice were monitored for mortality for 5 days. The functional activity of anti-OprF IgY was determined by in vitro invasion assays. Immunotherapy with anti-OprF IgY resulted in a significant improvement in the survival of mice infected by P. aeruginosa from 25% to 87.5% compared with the C-IgY and PBS. The anti-OprF IgY decreased the invasion of P. aeruginosa PAO1 into the A549. Passive immunization with anti-OprF IgY led to an efficacious protection against P. aeruginosa burn infection in the burn model.

A trivalent vaccine consisting of "flagellin A+B and pilin" protects against Pseudomonas aeruginosa infection in a murine burn model.

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly achieves antibiotic resistance, and thus, developing an effective vaccine is critically important for combating P. aeruginosa infection. Flagella and pili play important roles in colonization of P. aeruginosa at the burn wound site and its subsequent dissemination to deeper tissue and organs. In the present study, we evaluated protective efficacy of a trivalent vaccine containing flagellins A and B (FlaA + FlaB) + pilin (PilA) in a murine burn model of infection. "FlaA + FlaB + PilA" induced greater protection in P. aeruginosa murine burn model than the single components alone, and it showed broad immune protection against P. aeruginosa strains. Immunization with "FlaA + FlaB + PilA" induced strong opsonophagocytic antibodies and resulted in reduced bacterial loads, systemic IL-12/IL-10 cytokine expression, and increased survival after challenge with three times lethal dose fifty (LD50) of P. eruginosa strains. Moreover, the protective efficacy of "FlaA + FlaB + PilA" vaccination was largely attributed to specific antibodies. Taken together, these data further confirm that the protective effects of "FlaA + FlaB + PilA" vaccine significantly enhance efficacy compared with antibodies against either mono or divalent antigen, and that the former broadens the coverage against P. eruginosa strains that express two of the three antigens.

Anti-PcrV IgY antibodies protect against Pseudomonas aeruginosa infection in both acute pneumonia and burn wound models.

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly acquires antibiotic resistance; thus, developing an effective therapeutic approach is the most promising strategy for combating infection. Type III secretion system (T3SS) translocates bacterial toxins into the cytosol of the targeted eukaryotic cells, which plays important roles in the virulence of P. aeruginosa infections in both acute pneumonia and burn wound models. The PcrV protein, a T3SS translocating protein, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In the present study, we evaluated the protective efficacy of chicken egg yolk antibodies (IgY) raised against recombinant PcrV (r-PcrV) in both acute pneumonia and burn wound models. R-PcrV protein was generated by expressing the pcrV gene (cloned in pET-28a vector) in E. coli BL-21. Anti-PcrV IgY was obtained by immunization of hen. Anti-PcrV IgY induced greater protection in P. aeruginosamurine acute pneumonia and burn wound models than control IgY (C-IgY) and PBS groups. Anti-PcrV IgY improved opsonophagocytic killing and inhibition of bacterial invasion of host cells. Taken together, our data provide evidence that anti-PcrV IgY can be a promising therapeutic candidate for combating P. aeruginosa infections.

Molecular Identification of Cryptosporidium spp. in Iranian Dogs Using Seminested PCR: A First Report.

Cryptosporidium is a parasitic protozoon with a wide range of vertebrate hosts. Cryptosporidiosis has been reported from numerous countries, including Iran. Molecular identification can be applied to characterize Cryptosporidium, of which there are over 30 species and 50 genotypes. Herein, we report the genetic diversity of Cryptosporidium spp. in Iranian dogs for the first time based on 18S ribosomal RNA gene sequencing. One hundred forty fecal samples of herd dogs were collected from Isfahan, central Iran. The samples were concentrated using sucrose flotation and subjected to Kinyoun staining. DNA extraction of positive samples was performed, and molecular diagnosis was carried out using highly specific seminested PCR for the characterization of Cryptosporidium species. Finally, sequencing and DNA analysis were performed to identify Cryptosporidium species. A total of 2.14% of herd dogs were positive for cryptosporidiosis in both microscopy and molecular methods. In all cases, the causative agent was identified as Cryptosporidium parvum. Dogs associated with positive samples had been in close relationship with livestock. Cryptosporidiosis in the herd dogs in Isfahan could be due to their close contact with animals, particularly cattle and sheep. Given that dogs with cryptosporidiosis lack clinical symptoms, they are a potential source of zoonotic transmission of this disease as they are companion animals for humans. Dogs with cryptosporidiosis are a potential source of the zoonotic transmission of this disease.

Immunization with Bivalent Flagellin Protects Mice against Fatal Pseudomonas aeruginosa Pneumonia.

Pseudomonas aeruginosa lung infections present a major challenge to healthcare systems worldwide because they are commonly associated with high morbidity and mortality. Here, we demonstrate the protective efficacy of type a and b flagellins (bivalent flagellin) against acute fatal pneumonia in mice. Mice immunized intranasally with a bivalent flagellin vaccine were challenged by different flagellated strains of P. aeruginosa in an acute pneumonia model. Besides the protective effect of the vaccine, we further measured the host innate and cellular immunity responses. The immunized mice in our study were protected against both strains. Remarkably, active immunization with type a or b flagellin significantly improved survival of mice against heterologous strain compared to flagellin a or b antisera. We also showed that after an intranasal challenge by P. aeruginosa strain, neutrophils are recruited to the airways of vaccinated mice, and that the bivalent flagellin vaccine was proved to be protective by the generated CD4+IL-17+ Th17 cells. In conclusion, bivalent flagellin vaccine can confer protection against different strains of P. aeruginosa in an acute pneumonia mouse model by eliciting effective cellular and humoral immune responses, including increased IL-17 production and improved opsonophagocytic killing.

Bivalent flagellin immunotherapy protects mice against Pseudomonas aeruginosa infections in both acute pneumonia and burn wound models.

Pseudomonas aeruginosa infections are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics, thus renewing interest in antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Type a and b flagellins (flagellin a+b) of P. aeruginosa are acute virulence factors that play a major role in the establishment of infection. Here we evaluate the protective efficacy of antibodies raised against "flagellin a+b" in both acute pneumonia and burn models. A combination strategy using antibodies against "flagellin a+b" provided greater protection against cell invasion and enhanced opsono-phagocytosis and decreased motility of P. aeruginosa strains, compared to strategies using antibodies against a single flagellin. Antibodies against "flagellin a+b"-protected mice infected with P. aeruginosa strains significantly reduced bacterial dissemination from the site of infection to the liver and spleen. Passive immunization with antibodies against "flagellin a+b" led to an efficacious protection against P. aeruginosa infection in both acute pneumonia and burn models.

Antibodies raised against divalent type b flagellin and pilin provide effective immunotherapy against Pseudomonas aeruginosa infection of mice with burn wounds.

Burn wound infections caused by multidrug-resistant Pseudomonas aeruginosa strains are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics. Therefore, there is renewed interest in developing antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Here we used a murine model of burn wound infection to evaluate the efficacy of antibodies raised against the divalent type b flagellin and PilA (flagellin b + PilA), as acute virulence factors, to prevent and treat infection. Antibodies to flagellin b + PilA exhibited superior synergistic effects that improved opsono-phagocytosis and cell invasion compared with antibodies to each monovalent flagellin b or PilA. Further, when used for prophylaxis, the antibodies against flagellin b + PilA and combined therapeutic and prophylactic regimens markedly improved the survival of mice infected with disparate P. aeruginosa strains from 91.6% to 100% compared with treatment using imipenem. Therefore, antibodies against flagellin b + PilA interfere with the activities of their respective cognate individual target antigens and enhance coverage against clinical strains of P. aeruginosa that may not express one of these two virulence factors.

Passive immunization against Pseudomonas aeruginosa recombinant PilA in a murine burn wound model.

Pseudomonas aeruginosa type IV pili have an essential role in twitching motility, colonization and biofilm formation. In this study, we investigated the efficacy of intraperitoneal administration of rabbit anti-recombinant PilA (anti-r-PilA) immunoglobulin G (IgG) against P. aeruginosa infection in a mouse burn-wound model. After burn and infection, mortality rate was assessed in all mice, and that of mice passively immunized with rabbit anti-r-PilA IgG was compared to non-immunized mice. Bacterial quantities in the skin and internal organs were measured to determine the level of systemic infection. Results showed that passive immunotherapy with anti-r-PilA IgG protected the burned mice infected with P. aeruginosa strains, PAO1 and the clinical isolate (CI). Anti-r-PilA antibodies enhanced the opsonophagocytosis of these strains. Moreover, the administration of anti-r-PilA IgG was also successful in reducing the bacterial burden in infected mice. The reduction of systemic bacterial spread increased the survival rate of passively immunized mice. Findings of this study revealed an improved survival rate of 62.5%, thus confirming the protective effect of anti-r-PilA IgG.

Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody.

BACKGROUND: Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization. OBJECTIVES: As flagellin is the major component of the flagellar filament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin. MATERIALS AND METHODS: In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain by PCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera against the recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by in vitro assays. RESULTS: The polyclonal antibodies raised against this r-B-flagellin inhibited the motility of the homologous PAO1 strain of P. aeruginosa, which significantly decreased the invasion of the PAO1 strain into the A549 cells and also enhanced the opsonophagocytosis of this strain. However, our polyclonal antibody showed little effect on the heterologous PAK strain. CONCLUSIONS: The r-B-flagellin carried antigenic epitopes just like the native flagellin, while the polyclonal antibody raised against it exhibited functional activity.

Protective effect of pilin protein with alum+naloxone adjuvant against acute pulmonary Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an important opportunistic human pathogen that causes a wide variety of severe nosocomial infections. Type IV pili of P. aeruginosa are made up of polymerized pilin that aids in bacterial adhesion, biofilm formation and twitching motility. The aim of this study was to evaluate the efficacy of alum and naloxone (alum+NLX) as an adjuvant for P. aeruginosa recombinant PilA (r-PilA) as a vaccine candidate in the improvement of humoral and cellular immunity. Primary immunization with r-PilA in combination with alum+NLX followed by two booster shots was sufficient to generate robust cellular and humoral responses, which were Th1 and Th2 type responses consisting of IgG1 and IgG2a subtypes. Analysis of the cytokine response among immunized mice showed an increased production of IL-4, INF-γ and IL-17 by splenocytes upon stimulation by r-PilA. These sera were also able to reduce bacterial load in the lung tissue of challenged mice. The reduction of systemic bacterial spread resulted in increased survival rates in challenged immunized mice. In conclusion, immunization with r-PilA combined with alum+NLX evokes cellular and humoral immune responses, which play an important role in providing protection against acute P. aeruginosa lung infection among immunized mice.

Immunogenicity of Pseudomonas aeruginosa recombinant b-type fagellin as a vaccine candidate: Protective efficacy in a murine burn wound sepsis model.

Pseudomonas aeruginosa (PA) is a formidable opportunistic pathogen among patients with burn wound infections. Antimicrobial therapy is often unsuccessful because PA can develop multi-drug resistance; thus, immunotherapy can be a rational alternative. The goal of this study was to evaluate the immunogenicity recombinant type b flagellin (r-b-flagellin) as a potential vaccine against P. aeruginosa in a mouse model for burn wound sepsis. Primary immunization with r-b-flagellin (10µg) followed by two booster shots was sufficient to generate a robust humoral response, which was predominantly a T helper 2 (Th2) type response consisting mainly of subtype IgG1 and low levels of IgG2a. Analysis of the Th1-Th2 response among immunized mice showed an increased production of IL-4, INF-γ and IL-17 by splenocytes upon stimulation by r-b-flagellin. Opsono-phagocytosis assays confirmed the enhanced killing of bacteria by anti r-b-flagellin immune sera. These antibodies were also able to inhibit motility of P. aeruginosa and afforded protection to immunized mice by reducing bacterial load in the site of original infection into the liver of challenged mice. The reduction of systemic bacterial spread resulted in an increase in the survival rate of challenged immunized mice. In conclusion, immunization of mice with r-b-flagellin protein increased the level of humoral and cellular immune response and led to an efficacious protection against P. aeruginosa infection in the burn mouse model.

Flagellin and pilin immunization against multi-drug resistant Pseudomonas aeruginosa protects mice in the burn wound sepsis model.

Pseudomonas aeruginosa is a formidable pathogen and a major threat to burn patients. Antimicrobial therapy is often unsuccessful because P. aeruginosa can develop multi-drug resistance; thus, immunotherapy and vaccine can be a rational alternative. Flagella and type IV pili have been identified as important virulence factors in the colonization and pathogenesis of P. aeruginosa in burn wound infections. Immunogenicity and efficacy of mixed recombinant full-length type b flagellin (r-b-flagellin) and recombinant PilA (r-PilA) as candidate vaccines were assessed by measuring humoral and cellular responses, using an experimental burned mouse model. Primary immunization with "r-b-flagellin+r-PilA" followed by two booster shots was sufficient to generate a robust humoral response, which was predominantly a Th2 response consisting mainly of subtype IgG1 and low levels of IgG2a. Analysis of the cytokine response among immunized mice showed an increased production of IL-4, INF-γ and IL-17 by splenocytes upon stimulation by "r-b-flagellin+r-PilA". Opsonophagocytosis assays confirmed the enhanced killing of bacteria by anti "r-b-flagellin+r-PilA" immune sera. These antibodies were also able to reduce bacterial load in the site of original infection into the liver and spleen of challenged mice. The reduction of systemic bacterial spread resulted in an increased survival rate of challenged immunized mice. In conclusion, immunization with "r-b-flagellin+r-PilA" proteins provides a better protective response against P. aeruginosa infection in the burn mouse model.

Immunogenicity and protective efficacy of Pseudomonas aeruginosa type a and b flagellin vaccines in a burned mouse model.

Immunogenicity and efficacy of Pseudomonas aeruginosa type a and b flagellins (hereafter, flagellins) as candidate vaccines were evaluated using an experimental burned mouse model. The protection afforded and the reduction in bacterial burden achieved by these vaccine candidates were determined. Primary immunization with flagellins followed by two booster shots generated a robust immune response. Cytokine analysis demonstrated the secretion of interleukin-4 more than interferon-γ from immunized T-cells in response to in vitro antigen stimulation. IgG response was of Th2 type, predominantly with IgG1 and lower IgG2a levels before and after challenge. In vitro opsonophagocytosis assays confirmed protective potential of immune sera via enhanced bacterial cell killing. Immune sera also inhibited P. aeruginosa motility. Serum cytokine analysis demonstrated high IL-12 and low IL-10 levels in flagellin-immunized mouse sera. Reduced systemic bacterial spread from original infection site into liver and spleen was associated with increased survival. Immunization of mice with flagellins increased the humoral immune response and protection against P. aeruginosa infection in our mouse model.

Helicobacter pylori Infection and Dietary Factors Act Synergistically to Promote Gastric Cancer.

However, the incidence of gastric cancer (GC) has been decreased in past decades; GC is the second cause of cancer related death in the world. Evidence has illustrated that several factors including Helicobacter pylori (H. pylori) infection, host genetics, and environmental factors (smoking and particularly diet) may play a crucial role in gastric carcinogenesis. It has been demonstrated that high consumption of fresh fruits, vegetables, high level of selenium and zinc in drinking water, sufficient iron, and cholesterol protect against GC, while; smoked , pickled, and preserved foods in salt, and nitrites increase the risk of GC. Epidemiological studies have also proved that H. pylori infection and a high salt diet could independently induce atrophic gastritis and intestinal metaplasia. Recently, studies have been demonstrated that dietary factors directly influence H. pylori virulence. The use of appropriate diet could reduce levels of H. pylori colonization or virulence and prevent or delay development of peptic ulcers or gastric carcinoma. This is attractive from a number of perspectives including those of cost, treatment tolerability, and cultural acceptability. This review will describe new insights into the pathogenesis of H. pylori in relation to environmental factors, especially dietary, not only to find the developed means for preventing and treating GC, but also for understanding the role of chronic inflammation in the development of other malignancies.

Active Immunization with Recombinant PilA protein Protects Against Pseudomonas aeruginosa Infection in a Mouse Burn Wound Model.

Pseudomonas aeruginosa as an opportunistic human pathogen that causes lethal infections in immunocompromised patients. Type IV pili are critical factors in virulence and colonization of P. aeruginosa in acute burn wound infection. The immunogenicity and efficacy of P. aeruginosa recombinant PilA (r-PilA) was evaluated in an experimental model of burn wound sepsis as a vaccine candidate. In this study, female C57BL/6 mice were divided into five groups. Mice in the experimental groups received either r-PilA vaccine alone or in combination with the alum adjuvant or complete Freund's adjuvant (CFA). Mice in the negative control group received phosphate-buffered saline (PBS). In order to characterize the response of Th1-Th2 to immunization, the cytokine profiles of spleen cells isolated from r-PilA immunized mice were investigated. Total IgG titers and isotopes were measured using ELISA method and finally, in order to study the systemic infection, bacterial titers in the liver, spleen and blood were also determined. Active immunization with r-PilA, which is followed by two booster shots, was sufficient to generate a robust immune response in mice. Cytokine analysis demonstrated the secretion of IL-4 and INF-É£ from splenocytes in response to in vitro antigen stimulation. The IgG response to r-PilA was a Th2 type response consis¬¬ting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. In conclusion, in this burned mouse model, vaccination with r-PilA can increase the humoral immunity, thereby leading to an effective protection against P. aeruginosa infection.