Comparative Evaluation of Immune Responses and Protection of Chitosan Nanoparticles and Oil-Emulsion Adjuvants in Avian Coronavirus Inactivated Vaccines in Chickens.

Efficient vaccines are the main strategy to control the avian coronavirus (AvCoV), although several drawbacks related to traditional attenuated and inactivated vaccines have been reported. These counterpoints highlight the importance of developing new alternative vaccines against AvCoV, especially those able to induce long-lasting immune responses. This study evaluated and compared two inactivated vaccines formulated with AvCoV BR-I variants, one composed of chitosan nanoparticles (AvCoV-CS) and the second by Montanide oily adjuvant (AvCoV-O). Both developed vaccines were administered in a single dose or associated with the traditional Mass attenuated vaccine. The AvCoV-CS vaccine administered alone or associated with the Mass vaccine was able to induce strong humoral and cell-mediated immune (CMI) responses and complete protection against IBV virulent infection, wherein single administration was characterized by high IgA antibody levels in the mucosa, whereas when associated with the Mass vaccine, the serum IgG antibody was predominantly observed. On the other hand, single administration of the oily vaccine presented poor humoral and CMI responses and consequently incomplete protection against virulent challenge, but when associated with the Mass vaccine, immune responses were developed, and complete protection against infection was observed. Both of our experimental vaccines were able to induce full protection against virulent IBV challenge. A single dose of AvCoV-CS vaccine was sufficient to achieve complete protection, while AvCoV-O required a previous priming by a Mass strain to complete the protection.

Development of an indirect ELISA assay to evaluation of the adaptive immune response of pacu (Piaractus mesopotamicus).

The pacu is one of the most important species for Brazilian fish farming and is considered emerging in the global aquaculture. Despite its importance, no effective tool for evaluation of the adaptive immune response of this species has been developed. Therefore, this study aimed the development and standardization of indirect ELISA for the measurement of pacu antigen-specific antibodies using polyclonal rabbit anti-pacu IgM used as detector antibody. For this purpose was isolated and purificated of pacu IgM using mannose-binding protein affinity chromatography and produced specific polyclonal antibodies against heavy and light chains pacu IgM, that showed a molecular weight of 72 kDa and 26 kDa, respectively. Polyclonal antibodies obtained demonstrated specificity with heavy and light Ig chains of pacu serum in western blotting. These polyclonal antibodies allowed the development of an indirect ELISA assay of high sensitivity and specificity for the detection and quantification of pacu IgM antibodies immunized with bovine IgG. In conclusion, this approach has great potential to improve the monitoring of vaccine-induced immune responses and help develop immunodiagnostic and epidemiological studies of infectious diseases in pacu systems.

Assessment of molecular and genetic evolution, antigenicity and virulence properties during the persistence of the infectious bronchitis virus in broiler breeders.

The infectious bronchitis virus (IBV) causes a highly contagious disease [infectious bronchitis (IB)] that results in substantial economic losses to the poultry industry worldwide. We conducted a molecular and phylogenetic analysis of the S1 gene of Brazilian (BR) IBV isolates from a routinely vaccinated commercial flock of broiler breeders, obtained from clinical IB episodes that occurred in 24-, 46- and 62-week-old chickens. We also characterized the antigenicity, pathogenesis, tissue tropism and spreading of three IBV isolates by experimental infection of specific pathogen-free (SPF) chickens and contact sentinel birds. The results reveal that the three IBV isolates mainly exhibited mutations in the hypervariable regions (HVRs) of the S1 gene and protein, but were phylogenetically and serologically closely related, belonging to lineage 11 of the GI genotype, the former BR genotype I. All three isolates caused persistent infection in broiler breeders reared in the field, despite high systemic anti-IBV antibody titres, and exhibited tropism and pathogenicity for the trachea and kidney after experimental infection in SPF chickens and contact birds. In conclusion, BR genotype I isolates of IBV evolve continuously during the productive cycle of persistently infected broiler breeders, causing outbreaks that are not impaired by the current vaccination programme with Massachusetts vaccine strains. In addition, the genetic alterations in the S1 gene of these isolates were not able to change their tissue tropism and pathogenicity, but did seem to negatively influence the effectiveness of the host immune responses against these viruses, and favour viral persistence.

Expression of osteoblastic phenotype in periodontal ligament fibroblasts cultured in three-dimensional collagen gel.

OBJECTIVE: To investigate the influence of a three-dimensional cell culture model on the expression of osteoblastic phenotype in human periodontal ligament fibroblast (hPDLF) cultures. MATERIAL AND METHODS: hPDLF were seeded on bi-dimensional (2D) and three-dimensional (3D) collagen type I (experimental groups) and and on a plastic coverslip (control) for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity were performed. Also, cell morphology and immunolabeling for alkaline phosphatase (ALP) and osteopontin (OPN) were assessed by epifluorescence and confocal microscopy. The expression of osteogenic markers, including alkaline phosphatase, osteopontin, osteocalcin (OC), collagen I (COL I) and runt-related transcription factor 2 (RUNX2), were analyzed using real-time polymerase chain reaction (RT-PCR). Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. RESULTS: Experimental cultures produced an increase in cell proliferation. Immunolabeling for OPN and ALP in hPDLF were increased and ALP activity was inhibited by three-dimensional conditions. OPN and RUNX2 gene expression was significantly higher on 3D culture when compared with control surface. Moreover, ALP and COL I gene expression were significantly higher in three-dimensional collagen than in 2D cultures at 7 days. However, at 14 days, 3D cultures exhibited ALP and COL I gene expression significantly lower than the control, and the COL I gene expression was also significantly lower in 3D than in 2D cultures. Significant calcium mineralization was detected and quantified by alizarin red assay, and calcified nodule formation was not affected by tridimensionality. CONCLUSION: This study suggests that the 3D cultures are able to support hPDLF proliferation and favor the differentiation and mineralized matrix formation, which may be a potential periodontal regenerative therapy.

Expression of osteoblastic phenotype in periodontal ligament fibroblasts cultured in three-dimensional collagen gel

Objective : To investigate the influence of a three-dimensional cell culture model on the expression of osteoblastic phenotype in human periodontal ligament fibroblast (hPDLF) cultures. Material and Methods : hPDLF were seeded on bi-dimensional (2D) and three-dimensional (3D) collagen type I (experimental groups) and and on a plastic coverslip (control) for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity were performed. Also, cell morphology and immunolabeling for alkaline phosphatase (ALP) and osteopontin (OPN) were assessed by epifluorescence and confocal microscopy. The expression of osteogenic markers, including alkaline phosphatase, osteopontin, osteocalcin (OC), collagen I (COL I) and runt-related transcription factor 2 (RUNX2), were analyzed using real-time polymerase chain reaction (RT-PCR). Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Results : Experimental cultures produced an increase in cell proliferation. Immunolabeling for OPN and ALP in hPDLF were increased and ALP activity was inhibited by three-dimensional conditions. OPN and RUNX2 gene expression was significantly higher on 3D culture when compared with control surface. Moreover, ALP and COL I gene expression were significantly higher in three-dimensional collagen than in 2D cultures at 7 days. However, at 14 days, 3D cultures exhibited ALP and COL I gene expression significantly lower than the control, and the COL I gene expression was also significantly lower in 3D than in 2D cultures. Significant calcium mineralization was detected and quantified by alizarin red assay, and calcified nodule formation was not affected by tridimensionality. Conclusion : This study suggests that the 3D cultures are able to support hPDLF proliferation and favor the differentiation and mineralized matrix formation, which may be a potential periodontal regenerative therapy. .

Effects of electroacupuncture on experimental periodontitis in rats.

BACKGROUND: Acupuncture has shown the capability of modulating the immuno-inflammatory response of the host. This study aims to evaluate the effects of electroacupuncture (EA) on ligature-induced periodontitis in rats. METHODS: Thirty-two animals were divided into four groups: 1) control; 2) experimental periodontitis (EP); 3) sham-treated (EP/EA-sham); and 4) treated with EA (EP/EA). For the EP groups, a ligature was placed around the right mandibular first molars at day 1. Sessions of EA or EA-sham were assigned every other day. For EA treatment, large intestine meridian points LI4 and LI11 and stomach meridian points ST36 and ST44 were used. EA-sham was performed in off-meridian points. Animals were euthanized at day 11. Histomorphometric and microtomographic analyses were performed. Immunolabeling patterns for the receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) were assessed. Expressions of interleukin (IL)-1ß, matrix metalloproteinase (MMP)-8, IL-6, and cyclooxygenase (COX)-2 messenger RNAs (mRNAs) were evaluated by quantitative reverse transcription-polymerase chain reaction. Data were analyzed statistically (P <0.05, analysis of variance). RESULTS: Histomorphometric and microtomographic analyses demonstrated that group EP/EA presented reduced alveolar bone loss when compared to group EP (P <0.05). Reduced RANKL immunolabeling and fewer TRAP-positive multinucleated cells were observed in the EA-treated group in relation to group EP. No differences were observed in OPG expression among groups. EA treatment decreased the genic expression of IL-1ß and MMP-8 (P <0.05), increased the mRNA expression of IL-6 (P <0.05), and did not modify the genic expression of COX-2 in animals with EP (P >0.05). CONCLUSION: It can be concluded that EA reduced periodontal tissue breakdown and the expression of some proinflammatory mediators and a proresorptive factor in EP in rats.

Short-term evaluation of grafts fixed with either N-butyl-2-cyanocrylate or screws.

PURPOSE: N-butyl-2-cyanoacrylate (NB-Cn) is an alternative method for onlay graft fixation and might be efficient for preserving the graft volume. Our aim was to analyze the gene expression and mineralized tissue variations of calvarial bone grafting fixed in the mandible with either NB-Cn or a titanium screw (TiS). MATERIALS AND METHODS: New Zealand rabbits had bilateral calvarial grafts fixed at both sides of the mandible with either NB-Cn or a TiS. The rabbits were sacrificed at 4 and 8 days, and micro-computed tomography analysis was performed. For molecular analysis, the gene expression of interleukin-6, interleukin-10, and tumor necrosis factor-α was assessed. Quantification using real-time polymerase chain reaction was performed. Statistical analysis was performed using the paired Student t test (P < .05). RESULTS: Bone graft fixation with NB-Cn promoted superior volume and density preservation. The percentage of mineralized tissue at the center portion and border of the graft was very similar (NB-Cn, 50.6% ± 8.3% and 50.3% ± 10.6%, respectively) and superior than in the TiS group (32.5% ± 3.5% and 33.8% ± 6%, respectively). Genes from the NB-Cn group were upregulated compared with those in the TiS group at the initial phases of bone healing (4 days), with the profile reversed at the 8-day point. At day 8, the osteoclastogenesis-related genes were upregulated in the TiS group. CONCLUSIONS: Onlay bone grafts fixed with screws induced more inflammation during the initial remodeling process than did NB-Cn. The differences in the incorporation into the host bed suggest that the use of adhesives for graft fixation will promote superior volume and density preservation.

Periodontal disease: a genetic perspective.

Periodontitis is a multifactorial disease that causes tooth loss. The complex pathogenesis of periodontitis implies the involvement of a susceptible host and a bacterial challenge. Many studies have provided a valuable contribution to understanding the genetic basis of periodontal disease, but the specific candidate genes of susceptibility are still unknown. In fact, genome-wide studies and screening of single-nucleotide polymorphisms have yielded new genetic information without a definitive solution for the management of periodontal disease. In this manuscript, we provide an overview of the most relevant literature, presenting the main concepts and insights of the strategies that have been emerging to better diagnose and treat periodontal disease based on biomarker analysis and host modulation.

Periodontal disease: a genetic perspective

Periodontitis is a multifactorial disease that causes tooth loss. The complex pathogenesis of periodontitis implies the involvement of a susceptible host and a bacterial challenge. Many studies have provided a valuable contribution to understanding the genetic basis of periodontal disease, but the specific candidate genes of susceptibility are still unknown. In fact, genome-wide studies and screening of single-nucleotide polymorphisms have yielded new genetic information without a definitive solution for the management of periodontal disease. In this manuscript, we provide an overview of the most relevant literature, presenting the main concepts and insights of the strategies that have been emerging to better diagnose and treat periodontal disease based on biomarker analysis and host modulation.

An experimental model of meningoencephalomyelitis by Rocio flavivirus in BALB/c mice: inflammatory response, cytokine production, and histopathology.

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients.

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients / Avaliação de genótipos de glicoproteína B e carga de CMV de leucócitos no prognóstico de pacientes de AIDS

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31 percent) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9 percent). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16 percent) followed by gB3 (35.48 percent). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80 percent) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.

ANTECEDENTES: O citomegalovírus (CMV) permanece um importante patógeno para pacientes imunocomprometidos, mesmo na era da HAART. O presente estudo teve como objetivo avaliar a influência da carga viral do CMV e seu genótipo gB sobre a evolução de pacientes com AIDS. MÉTODOS: Amostras de sangue de 101 pacientes com AIDS foram coletadas e testadas para carga viral de HIV, a contagem de células CD4 e patógenos oportunistas, incluindo o CMV. Um sistema de PCRs seminested foi utilizado para detectar o genoma do CMV e em amostras positivas a carga viral de CMV e genotipagem foram estabelecidos por restrição enzimática e PCR em tempo real, respectivamente. Todos os pacientes foram acompanhados clinicamente durante quatro anos. RESULTADOS: Trinta pacientes (31 por cento) tiveram CMV detectado e todos os casos fatais (n = 5) ocorreram em pacientes deste grupo (p = 0,007), porém apenas dois pacientes tinham doença por CMV (1,9 por cento). No entanto, a carga viral não foi associada estatisticamente a nenhum dos parâmetros analisados. O genótipo de CMV mais freqüentemente observado foi gB2 (45,16 por cento), seguido por gB3 (35,48 por cento). O genótipo gB2 foi mais freqüente em pacientes com contagens abaixo de 200 células/mm³ CD4cell (p = 0,0017), e quase todos os casos fatais (80 por cento) tinham o genótipo gB2. CONCLUSÃO: Nosso estudo sugere que CMV e seu polimorfismo em genes relevantes biologicamentes, como a gB, pode ainda influenciar no prognóstico e evolução de pacientes com AIDS. O genótipo gB2 foi associado ao mau prognóstico do paciente.

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis.

Cytomegalovirus (CMV) is a genus in the family Herpesviridae that has been associated with gastrointestinal syndromes. In this work we looked for a possible association of CMV infection with colorectal cancer and ulcerative colitis (UC). Blood and enteric tissue samples of 14 patients with colorectal cancer and of 21 with UC were subjected to a nested-PCR that amplifies part of the gB gene of CMV and also to immunohistochemistry using a specific monoclonal antibody to IE 76 kDa protein of CMV. CMV was detected by nested-PCR in the blood and/or the enteric tissue of nine (64.3%) colorectal cancer and 16 (76.2%) ulcerative colitis patients. In the immunohistochemistry it was observed that 12 (12/21, 57.1%) positive enteric tissue samples of patients with UC and none from patients with colorectal cancer (0/14) were positive to CMV. The positivity of CMV infections in the UC patient group (12/21, 57.1%) showed by both techniques, was significantly higher (p = 0.015) than that observed for colorectal cancer patients (2/14, 14.3%). These results suggest an association of ulcerative colitis with CMV infection of the enteric tissue.

Cytomegalovirus in colorectal cancer and idiopathic ulcerative colitis

Cytomegalovirus (CMV) is a genus in the family Herpesviridae that has been associated with gastrointestinal syndromes. In this work we looked for a possible association of CMV infection with colorectal cancer and ulcerative colitis (UC). Blood and enteric tissue samples of 14 patients with colorectal cancer and of 21 with UC were subjected to a nested-PCR that amplifies part of the gB gene of CMV and also to immunohistochemistry using a specific monoclonal antibody to IE 76kDa protein of CMV. CMV was detected by nested-PCR in the blood and/or the enteric tissue of nine (64.3 percent) colorectal cancer and 16 (76.2 percent) ulcerative colitis patients. In the immunohistochemistry it was observed that 12 (12/21, 57.1 percent) positive enteric tissue samples of patients with UC and none from patients with colorectal cancer (0/14) were positive to CMV. The positivity of CMV infections in the UC patient group (12/21, 57.1 percent) showed by both techniques, was significantly higher (p = 0.015) than that observed for colorectal cancer patients (2/14, 14.3 percent). These results suggest an association of ulcerative colitis with CMV infection of the enteric tissue.

Os Cytomegalovírus (CMV) são um gênero da família Herpesviridae, que pode estar associado a síndromes gastrointestinais. No presente trabalho buscamos uma possível associação da infecção por CMV com câncer coloretal e retocolite ulcerativa (RCU). Amostras de sangue e tecido entérico de 14 pacientes com câncer coloretal e 21 com RCU foram submetidas a uma nested-PCR que amplifica parte do gene gB do CMV e a uma imunohistoquímica utilizando um anticorpo monoclonal específico para proteína IE 76Kda de CMV. CMV foi detectado pela nested-PCR em sangue e/ou tecido entérico de 9 (64,3 por cento) dos pacientes com câncer coloretal e 16 (76,2 por cento) dos pacientes com RCU. Na imunohistoquímica foram observadas 12 (57,1 por cento) amostras positivas para CMV nos pacientes com RCU e nos pacientes com câncer coloretal o CMV não foi detectado em nenhuma amostra. A positividade das infecções no grupo de pacientes com RCU (12/21, 57.1 por cento) foi significantemente mais alta (p = 0,015) que aquela observada nos pacientes com câncer coloretal (2/14, 14.3 por cento). Estes resultados sugerem uma associação da presença de CMV no tecido entérico com RCU.