[Experimental research on Arginine-gingipain A gene vaccine from Porphyromonas gingivalis that prevents peri-implantitis in Beagle dogs].

OBJECTIVE: This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development. METHODS: Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 µm were prepared and dyed with methylene blue to observe the bone phenotype around implants. RESULTS: Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P<0.05) and higher than those in group C (P<0.05). However, no difference was observed between groups A and B (P>0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P<0.05). On the other hand, no difference was identified between groups A and B (P>0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants. CONCLUSIONS: IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.

Dinámica estructural de colonias/biofilm de Legionella pneumophila y Legionella bozemanii / Structural dynamics of Legionella pneumophila and Legionella bozemanii colony/biofilm

Objetivos. El género Legionella engloba especies muy pleomórficas responsables de brotes infecciosos en humanos. En la aparición de los mismos tiene gran importancia el desarrollo de biofilms en ecosistemas acuáticos artificiales. El objetivo de este trabajo fue estudiar la dinámica de crecimiento y la evolución de la estructura interna de colonias de especies representativas del género como modelo de biofilm estático. Material y métodos. Colonias aisladas de Legionella pneumophila y Legionella bozemanii crecidas en medios específicos durante tres y quince días fueron procesadas por métodos histológicos de inclusión en parafina y resina epoxi para su análisis mediante microscopía óptica, microscopía electrónica y análisis de imagen. Resultados. En las colonias de ambas especies se observaron patrones arquitecturales definidos y específicos, basados en la estratificación y que evolucionan en el tiempo. Los estratos se diferencian por la cantidad de matriz extracelular, la morfología y densidad poblacional y la proporción de células muertas. La estructura interna de las colonias de tres días presentaba grandes diferencias entre L. pneumophila (dos estratos) y L. bozemanii (cuatro estratos). Sin embargo, en las colonias de quince días ambas especies evolucionaron hacia un patrón único común formado por tres estratos. En ambas especies se comprobó también el crecimiento en el interior del medio de cultivo, aunque este fenómeno fue mucho más intenso en L. bozemanii, con invasiones únicas, centrales y de gran tamaño. Conclusiones. Nuestros resultados demuestran que las colonias de Legionella sobre medio de cultivo sólido son un buen modelo de biofilm estático, con una dinámica estructural compleja caracterizada por la presencia de subpoblaciones morfológicas y funcionales. La aproximación histológica empleada en este modelo permitirá estudiar adaptaciones evolutivas de comunidades multicelulares a medios hostiles, así como la respuesta a los antimicrobianos de las especies de Legionella de interés clínico (AU)

Objectives. The genus Legionella includes very pleomorphic species responsible for disease outbreaks in humans. The appearance of such has great importance to develop artificial biofilms in aquatic ecosystems. The aim of this work was to study the dynamics of growth and evolution of the internal structure of colonies of representative species of the genus as static biofilm model. Methods. Isolated colonies of Legionella pneumophila and Legionella bozemanii grown in specific media for three and fifteen days were processed for histological methods and embedded in paraffin and epoxy resin for analysis by light microscopy, electron microscopy and image analysis. Results. In colonies of both species were observed and defined specific architectural patterns, based on stratification and evolve over time. The strata differ in the amount of extracellular matrix, the morphology and population density and the proportion of dead cells. The internal structure of three days colonies showed large differences between L. pneumophila (two layers) and L. bozemanii (four layers). However, in the fifteen days colonies of both species evolved towards a common unique pattern formed by three layers. In both species the growth was also found within the culture medium, although this phenomenon was more intense in L. bozemanii with unique, central and larger invasions. Conclusions. Our results demonstrate that Legionella colonies on solid culture media are a good model of static biofilm with a complex structural dynamics characterized by the presence of morphological and functional subpopulations. We bring here an histological approach model, allowing, in further research, detailed studies in evolutionary adaptations in multicellular communities to adverse media and to antimicrobials in Legionella species of clinical interest (AU)

Combat pneumococcal infections: adhesins as candidates for protein-based vaccine development.

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the upper respiratory tract in humans. However, these apparently harmless bacteria have also a high virulence potential and are known as the etiologic agent of respiratory and life-threatening invasive diseases. Dissemination of pneumococci from the nasopharynx into the lungs or bloodstream leads to community-acquired pneumonia, septicaemia and meningitis. Traditionally, pneumococcal diseases are treated with antibiotics and prevented with polysaccharide-based vaccines. However, due to the dramatic increase in antibiotic resistance and limitations of the current available vaccines, the burden of diseases remains high. Thus, combating pneumococcal transmission and infections has emphasized the need for a new generation of protein-based vaccines. Interactions of pneumococci with soluble host proteins or cellular receptors are crucial for adherence, colonization, transmigration of host barriers and immune evasion. Therefore, surface-exposed proteins involved in these pathogenic processes and virtually expressed by all pneumococcal strains and serotypes are the prime potential targets for an immunogenic and highly protective pneumococcal-derived carrier protein of a vaccine. In this review, we will address the state of the art in deciphering, i). the conservation, distribution and pathogenic role of recently discovered pneumococcal adhesins in colonization and invasive diseases, ii). the interactions of these virulence factors with host-proteins and receptors, iii). the subversion of the host immune and cellular responses, and iv). the potential of pneumococcal adhesins as vaccine candidates.

Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomodulatory molecule.

BACKGROUND: Filamentous haemagglutinin (FHA) of Bordetella pertussis subverts host immune responses by inhibiting interleukin (IL)12 and enhancing IL10 production by macrophages and dendritic cells, and promoting the induction of regulatory T cells. HYPOTHESIS: Injection of FHA would ameliorate disease in a T cell-dependent model of colitis via the induction of anti-inflammatory cytokines and regulatory T cells. METHODS: Colitis was induced by injection of CD4CD45RB(high) naive T cells into severe combined immunodeficient (SCID) mice. Mice were treated with four subcutaneous injections of FHA or buffer alone. RESULTS: Parenteral injection of FHA stimulated IL10 and/or transforming growth factor beta production in local and mesenteric lymph nodes and Peyer's patches of mice 2-6 h after administration. Compared with phosphate-buffered saline-treated mice, FHA-treated SCID mice had significantly (p<0.01) less weight loss, lower colon weight, less colon shrinkage and reduced inflammatory lesions. The therapeutic effect of FHA was associated with enhanced IL10 and reduced type 1 and type 2 T helper cytokine production by spleen cells. Finally, FHA also attenuated the symptoms of colitis in SCID mice transferred with CD4CD45RB(high) T cells from IL10-deficient mice. CONCLUSIONS: Our finding shows that FHA suppresses type 1 T helper and pro-inflammatory cytokines, and ameliorates disease activity in a chronic T cell-dependent model of colitis, an effect that was not dependent on IL10 production by T cells, but was associated with induction of anti-inflammatory cytokines in vivo. Having already been used as a pertussis vaccine component in children, FHA is a promising candidate for clinical testing in patients with Crohn's disease.

Current progress of adhesins as vaccine candidates for Moraxella catarrhalis.

Moraxella catarrhalis is an emerging pathogen and all isolates are now considered beta-lactamase producing. Potential further use of vaccines against Streptococcus pneumoniae and nontypeable Haemophilus influenzae means that M. catarrhalis might be thrust further into the limelight. However, a vaccine has not yet been designed. In this review, the progress of M. catarrhalis adhesins as vaccine candidates is discussed with a focus on various candidate antigens that spanned those discovered more than 10 years ago, for example, the ubiquitous surface proteins to newer antigens, such as the Moraxella IgD-binding hemagglutinin.

An immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis-induced periodontal bone loss.

Porphyromonas gingivalis, a pathogen associated with periodontitis, bound to fibrinogen, fibronectin, hemoglobin, and collagen type V with a similar profile to that of its major virulence factor, the cell surface RgpA-Kgp proteinase-adhesin complex. Using peptide-specific, purified Abs in competitive inhibition ELISAs and epitope mapping assays, we have identified potential adhesin binding motifs (ABMs) of the RgpA-Kgp complex responsible for binding to host proteins. The RgpA-Kgp complex and synthetic ABM and proteinase active site peptides conjugated to diphtheria toxoid, when used as vaccines, protected against P. gingivalis-induced periodontal bone loss in the murine periodontitis model. The most efficacious peptide and protein vaccines were found to induce a high-titer IgG1 Ab response. Furthermore, mice protected in the lesion and periodontitis models had a predominant P. gingivalis-specific IL-4 response, whereas mice with disease had a predominant IFN-gamma response. The peptide-specific Abs directed to the ABM2 sequence (EGLATATTFEEDGVA) protected against periodontal bone loss and inhibited binding of the RgpA-Kgp complex to fibrinogen, fibronectin, and collagen type V. Furthermore, the peptide-specific Abs directed to the ABM3 sequence (GTPNPNPNPNPNPNPGT) protected against periodontal bone loss and inhibited binding to hemoglobin. However, the most protective Abs were those directed to the active sites of the RgpA and Kgp proteinases. The results suggest that when the RgpA-Kgp complex, or functional binding motif or active site peptides are used as a vaccine, they induce a Th2 response that blocks function of the RgpA-Kgp complex and protects against periodontal bone loss.

Immunogenicity of a three-component acellular pertussis vaccine administered at birth.

OBJECTIVE: To evaluate within the first 6 months of birth the immunogenicity of a 3-component acellular pertussis (aP) vaccine containing filamentous hemagglutinin (FHA), pertactine (PRN), and genetically detoxified pertussis toxin (PT) in infants who received a dose of vaccine at birth, in addition to the recommended schedule administered at 3, 5, and 11 months. Furthermore, we investigated the influence of maternal antibodies on aP vaccine response. METHODS: We used enzyme-linked immunosorbent assay to evaluate immunoglobulin G antibody levels in 45 infants immunized at birth and at 3, 5, and 11 months (group 1) and in 46 infants immunized at the ages of 3, 5, and 11 months (group 2). All mothers were also tested at delivery. RESULTS: At the age of 5 months the geometric mean titer of anti-PT, anti-FHA, and anti-PRN was significantly greater in group 1 (who had received 2 doses) than in group 2 (1 dose). At 6 months geometric mean titers were significantly higher in group 1 than in group 2 for anti-PRN and anti-FHA, whereas no significant differences were observed for anti-PT. CONCLUSIONS: Immunization at birth may be important for an earlier prevention of the pertussis disease in infants under 6 months, especially in Italy, where the recommended ages for aP vaccine administration are 3, 5, and 11 months.

Natural antibodies and their significance in active immunization and protection against a defined pathogen in fish.

Natural antibody activity against Aeromonas salmonicida extracellular A-layer protein (A-protein) showed large individual variations in a farmed group of 101 goldfish (Carassius auratus L.). Statistical analyses of these variations led us to divide this group into homogeneous high and low naturally active (HNA and LNA) subgroups. The HNA fish were largely protected against experimental infection with a virulent atypical A. salmonicida strain, while 100% morbidity was recorded in the LNA group. In the course of active immunization with a particulate form of A-protein, a significant antibody response was exhibited by the LNA group only. Significance and implication of these results in vaccination practice are discussed.

Cross-protective immunity of mice induced by oral immunization with pneumococcal surface adhesin a encapsulated in microspheres.

The global use of a capsular polysaccharide-based pneumococcal vaccine has been limited because of serotype-specific protection and poor effectiveness in individuals with low immunocompetency. The mucosal immune system develops earlier in infants and lasts longer in the elderly than does the systemic immune system. Furthermore, mucosal immunization is beneficial for AIDS patients, because human immunodeficiency virus-infected subjects can develop normal mucosal antibody responses even in late clinical phases. For these reasons, we evaluated recombinant pneumococcal surface adhesin A (rPsaA) of Streptococcus pneumoniae in terms of cross-protective immune responses after oral delivery. Encapsulated rPsaA provided higher immunogenicity than naked rPsaA. Coencapsulation or codelivery of the cholera toxin (CT) B subunit (CTB) and CT also increased the immunogenicity of rPsaA. Cross-protective immunities against five strains of S. pneumoniae (types 4, 6B, 14, 19F, and 23F) were induced after oral immunization with microencapsulated rPsaA. Lung colonization and septicemia caused by the five serotypes were significantly inhibited by oral immunization with microencapsulated rPsaA. These results suggest that rPsaA coencapsulated with CTB can be used as an oral vaccine to induce cross-protective immunity for the prevention of pneumococcal infection.

Synthetic peptide derived from the Bordetella pertussis bacterium reduces infarct volume after transient middle cerebral artery occlusion in the rat.

We explored the therapeutic potential of a peptide (F20) derived from the filamentous hemagglutinin of Bordetella pertussis in a model of ischemic cell injury after transient (2 hours) middle cerebral artery (MCA) occlusion in the rat. Animals were divided into two groups-(1) F20 peptide group: rats (n = 11) were subjected to 2 hours of transient MCA occlusion, and F20 peptide was administered intravenously (50 nmol) at 0 hours of reperfusion and intraperitoneally (150 nmol/dose) at 2, 4, 6, 8, 22, and 30 hours of reperfusion; (2) control group: rats (n = 10) were administered peptide F23 (a scrambled version of peptide F20) with the same experimental protocol as the F20 peptide group. Forty-six hours after reperfusion, animals were sacrificed, and brain tissue was stained with triphenyltetrazolium chloride for evaluation of tissue damage. To measure neutrophil numbers in ischemic tissue, myeloperoxidase (MPO) immunostaining was performed on a coronal cerebral section in each animal. There was a significant reduction of ischemic infarct volume (36%, p < 0.05) in the F20 group of animals compared with the F23 group. The area of the ischemic lesion was highly correlated with the numbers of the immunoreactive MPO cells (r = 0.78, p < 0.001). The data demonstrate that the F20 peptide significantly reduces infarct volume and intraparenchymal neutrophil numbers after transient MCA occlusion.