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. .

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.

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 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.