Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients

Background and Objective: Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT. Material and Methods: Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed. Results: Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits. Conclusion: Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in Sao Paulo, Brazil

Aims: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. Methods and Results: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. Conclusions: This study demonstrated that healthy sheep in Sao Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. Significance and Impact of the Study: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade I PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed. (C) 2008 Elsevier Masson SAS. All rights reserved.

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases.

Intervenção com pais de crianças deficientes auditivas: elaboração e avaliação de um programa de orientação não presencial

As orientações aos pais favorecem a aceitação da deficiência auditiva e esclarecem possibilidades e condutas que viabilizam o desenvolvimento da criança. Devem ser cuidadosas, a fim de evitar insegurança, ansiedade, expectativas irreais ou reações inadequadas dos pais. Essas orientações são oferecidas no diagnóstico e no acompanhamento, mas as restrições de tempo e financeiras são dificuldades dos pais para o comparecimento freqüente a programas que forneçam um suporte contínuo. A proposta deste trabalho foi elaborar e avaliar um programa de orientação não presencial para pais de crianças com deficiência auditiva severa e profunda, de dois a seis anos de idade. O programa, estruturado em quatro unidades, foi aplicado a 30 pais atendidos no Hospital de Reabilitação de Anomalias Craniofaciais da USP, em Bauru/SP. As unidades foram formuladas com base nas orientações que os especialistas de diferentes áreas transmitem durante o diagnóstico da deficiência auditiva e nas dificuldades e interesses dos pais, identificando-se o conhecimento sobre as avaliações e acompanhamentos, opiniões sobre desempenho e necessidades da criança, deles próprios e das famílias. Para avaliação do programa foram analisadas as respostas dos pais aos questionários das unidades e às entrevistas finais. As análises revelaram que o programa forneceu aos pais, como eles desejavam, informações claras e sugestões de atividades que pudessem ser adequadas ao contexto familiar e colaborassem para o desenvolvimento da criança. Alguns pais encontraram dificuldade em se expressar por escrito e realizar algumas atividades, mas nas entrevistas, foi verificado que tal fato não inviabilizou o entendimento e a participação no programa.

Cuidador familiar de seqüelados de acidente vascular cerebral: significado e implicações

O acidente vascular cerebral (AVC) está entre as principais causas de morte no mundo. Diante das incapacidades impostas pelo AVC, surge o cuidador familiar. O objetivo desta pesquisa foi analisar o significado e as implicações de se tornar cuidador de um indivíduo seqüelado de AVC. Trata-se de um estudo qualitativo, realizado com cinco cuidadores, em Londrina-PR, no período de agosto a dezembro de 2005. Os dados foram coletados por meio de entrevistas semi-estruturadas e submetidos à análise de discurso proposta por Martins e Bicudo. Da análise das entrevistas emergiram sete categorias: voltando-se para o Ser cuidado, descrevendo os cuidados, apresentando as mudanças, o apoio da espiritualidade, compreendendo o significado do cuidado, expressando as necessidades para cuidar e perspectivas para o futuro. Os resultados revelaram que ser cuidador familiar é um fenômeno complexo, que gera nos sujeitos sentimentos de alegria e felicidade concomitantes aos sentimentos de medo, ansiedade e revolta.

Simplified flow cytometric assay to detect minimal residual disease in childhood with acute lymphoblastic leukemia

The detection of minimal residual disease (MRD) is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL) providing crucial information on the response to treatment and risk of relapse. However, the high cost of these techniques restricts their use in countries with limited resources. Thus, we prospectively studied the use of flow cytometry (FC) with a simplified 3-color assay and a limited antibody panel to detect MRD in the bone marrow (BM) and peripheral blood (PB) of children with ALL. BM and PB samples from 40 children with ALL were analyzed on days (d) 14 and 28 during induction and in weeks 24-30 of maintenance therapy. Detectable MRD was defined as > 0.01% cells expressing the aberrant immunophenotype as characterized at diagnosis among total events in the sample. A total of 87% of the patients had an aberrant immunophenotype at diagnosis. On d14, 56% of the BM and 43% of the PB samples had detectable MRD. On d28, this decreased to 45% and 31%, respectively. The percentage of cells with the aberrant phenotype was similar in both BM and PB in T-ALL but about 10 times higher in the BM of patients with B-cell-precursor ALL. Moreover, MRD was detected in the BM of patients in complete morphological remission (44% on d14 and 39% on d28). MRD was not significantly associated to gender, age, initial white blood cell count or cell lineage. This FC assay is feasible, affordable and readily applicable to detect MRD in centers with limited resources.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Fracionamento dos carboidratos pelas equações do Cornell Net Carbohydrate and Protein System de três cultivares de girassol na presença ou não de irrigação

Objetivou-se quantificar as frações de carboidratos pelas equações do Cornell Net Carbohydrate and Protein System (CNCPS) de três cultivares de girassol (Helianthus annuus L.) cultivados na presença ou não de irrigação. A utilização de uma preparação fibrosa, denominada parede celular (PC), nas equações da CNCPS, em substituição à fibra em detergente neutro (FDN) não promoveu diferenças nas frações de carboidratos B1 e C, mas influenciou as frações A e B2. Como os valores da fração B1, obtidos pelo modelo CNCPS foram menores que os teores de amido e pectina determinados em laboratório, supõe-se que a pectina e outros oligossacarídeos da parede celular, solubilizados pela solução de detergente neutro (fibra solúvel), nunca fizeram parte da fração B1, e sim da fração A. Apesar de os carboidratos da fibra solúvel apresentarem elevadas taxas de degradação, não parece adequada a caracterização da fibra solúvel na fração A. Parece mais adequado que a fibra solúvel (que inclui a pectina) seja alocada a uma fração exclusivamente sua, que pode ser a fração B2, e que seja criada uma nova fração, a B3, para os carboidratos digeríveis da parede celular. Assim, a fração B1 seria composta apenas de amido. A equação da fração C, que estima os carboidratos indigeríveis da parede celular, pode ser simplificada, relacionando a fração indigerível ao teor de lignina na matéria seca, e não à FDN isenta de cinzas e proteína, como atualmente utilizado. Esta proposta tem implicações práticas, uma vez que a fração indigerível da parede celular tem sido expressa em relação à FDN, e não na MS, com base no fato de que os efeitos inibitórios da lignina ocorrem sobre os componentes fibrosos da parede celular vegetal, e não sobre o conteúdo celular.

Surveillance for adverse events after DTwP/Hib vaccination in Brazil: sensitivity and factors associated with reporting

We estimated the sensitivity, i.e., the proportion of all cases of adverse events following immunization (AEFIs) reported to the Brazilian passive surveillance for adverse events following immunization (PSAEFI) with the diphtheria-tetanus-whole-cell pertussis-Haemophilus influenzae type b (DTwP-Hib) vaccine, as well as investigating factors associated with AEFIs reporting. During 2003–2004, 8303 AEFIs associated with DTwP-Hib were reported; hypotonic-hyporesponsive episodes (HHEs), fever and convulsions being the most common. Cure without sequel was achieved in 98.4 per cent of the cases. The mean sensitivity of the PSAEFI was 22.3 per cent and 31.6 per cent, respectively, for HHE and convulsions, varying widely among states. Reporting rates correlated positively with the Human Development Index and coverage of adequate prenatal care, correlating negatively with infant mortality rates. Quality of life indicators and the degree of organization of health services are associated with greater PSAEFI sensitivity. In addition to consistently describing the principal AEFIs, PSAEFI showed the DTwP/Hib vaccine to be safe and allayed public fears related to its use

Aeromonas detection and their toxins from drinking water form reservoirs and drinking fountains

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in São Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A.allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%).The results revealed that 70% of A. caviae, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern

Bronchus-Associated Lymphoid Tissue (BALT) and Survival in a Vaccine Mouse Model of Tularemia

Background: Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant. Methodology/Principal Findings: Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas. Conclusions: These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.

ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser(1270)

Objectives: We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser(1270) are involved in shear-stress (SS)-induced downregulation of the enzyme. Methods and Results: Western blotting analysis showed that SS (18 h, 15 dyn/cm(2)) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra-or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser(1270) compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. Conclusions: ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser(1270), consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser(1270).