Platelet-rich plasma enhances the differentiation of dental pulp progenitor cells into odontoblasts.

AIM: To investigate the effects of PRP on odontoblastic differentiation using dental pulp progenitor cells derived from the dental papilla of rat incisors. METHODOLOGY: Monolayer cultures of odontoblastic lineage KN-3 cells were incubated with PRP for various time periods. The expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1) was determined using real-time reverse transcription-polymerase chain reaction and Western blot analyses. To further clarify the role of PRP in odontogenesis, KN-3 cells were stimulated with PRP in the presence of ascorbic acid and ß-glycerophosphate. The cells were stained for alkaline phosphatase (ALP), and ALP activity was quantified in cell lysates. The formation of mineralized nodules was assessed by alizarin red staining. Statistical analysis was performed by one-way analysis of variance. RESULTS: PRP increased the mRNA and protein expressions of odontoblastic markers, such as DSPP and DMP-1. Furthermore, PRP stimulated the ALP activity and mineralized nodule formation induced by ascorbic acid and ß-glycerophosphate in a time-dependent manner. CONCLUSIONS: PRP enhances odontoblastic differentiation of KN-3 cells. These results indicate that PRP could be a potential candidate for use in the regeneration of dentine-pulp complex.

Variety and complexity of fluorine-18-labelled fluoro-2-deoxy-D-glucose accumulations in the oral cavity of patients with oral cancers.

OBJECTIVES: To elucidate the points that require attention when interpreting fluorine-18-labelled fluoro-2-deoxy-d-glucose ((18)F-FDG)/positron emission tomography (PET) images by demonstration of (18)F-FDG accumulation in various areas of the oral cavity other than primary lesions in patients with oral cancers. METHODS: (18)F-FDG accumulations with a maximal standardized uptake value of over 2.5 in various areas of the oral cavity other than primary lesions were identified in 82 patients with oral cancers. RESULTS: (18)F-FDG/PET-positive areas, excluding primary tumours, included the front intrinsic muscles of the tongue (89.0%), upper and lower marginal parts of the orbicularis oris muscle (64.6%), sublingual glands, palatine tonsil, pharyngeal tonsil, and lingual tonsil. In addition, some areas in the jaws also showed accumulation. CONCLUSIONS: In patients with oral cancers, areas of (18)F-FDG accumulation in the oral cavity should be precisely identified and appropriately diagnosed, because accumulations can be seen in areas other than the primary tumour.

Sesamin suppresses activation of microglia and p44/42 MAPK pathway, which confers neuroprotection in rat intracerebral hemorrhage.

Thrombin plays important roles in the pathology of intracerebral hemorrhage (ICH). The recruitment of activated microglia, accompanied by thrombin-induced phosphorylation of the mitogen-activated protein kinase (MAPK) family, contributes to ICH-associated neuron loss. Here we investigated the possibility that sesamin, a lignan of sesame seed oil, is a natural candidate as an inhibitor of microglial activation and MAPK pathways under ICH insults. Sesamin (30-100 µM) suppressed thrombin-induced nitric oxide (NO) production by primary-cultured rat microglia via inhibition of inducible NO synthase (iNOS) protein expression, independently of the antioxidative effect. Sesamin selectively inhibited p44/42 MAPK phosphorylation in the MAPK family (p38 and p44/42) involved in iNOS protein expression in primary-cultured rat microglia. An in vivo rat ICH model was prepared by intrastriatal injection of 0.20U collagenase type IV unilaterally. ICH evoked the phosphorylation of p44/42 MAPK, microglial proliferation with morphological change into the activated ameboid form, and neuron loss. The phosphorylation of p44/42 MAPK was inhibited by intracerebroventricular administration of 30-nmol sesamin. Sesamin prevented ICH-induced increase of microglial cells in the perihematomal area. Notably, ramified microglia, the resting morphology, were observed in brain sections of the animals administrated sesamin. Sesamin furthermore achieved neuroprotection in the perihematomal area but not in the hematomal center. These results suggest that sesamin is a promising natural product as a novel therapeutic strategy based on the regulation of microglial activities accompanied by the activated p44/42 MAPK pathway in ICH.

How effective is video consultation in clinical oncology? A systematic review.

BACKGROUND: Video-consultation (VC) is a specialized type of telemedicine that uses technology to provide real-time visual and audio patient assessment at a distance. In the present review, we set out to evaluate whether vc is feasible for the assessment, monitoring, and management of oncology patients. METHODS: A search strategy designed to capture studies that addressed the use of telemedicine to deliver cancer care identified relevant articles in the medline (1966 to September 2008) and PubMed (to 2008) databases. Articles were included if they described studies incorporating video-conferencing between patient and provider for assessment or monitoring,physicians or nurses as the care providers,cancer patients,consultation in real-time, and reporting of 1 or more outcomes. RESULTS: Of the more than three hundred articles retrieved, nineteen articles describing 15 unique patient populations involving 709 patients were inclusded in the analysis. No randomized trials were located. Eight studies included a control group; seven involved a case series. The most commonly reported outcomes were patient satisfaction (ten studies), cost to perform consultation (six studies), patient preference for vc compared with in-person consultation (five studies), provider satisfaction (four studies), and provider convenience (four studies). Of these outcomes, satisfaction on the part of patients and physicians has been positive overall, total costs were comparable to or less than those for in-person consultations, and patients valued having vc as an option for consultation. Outcomes evaluating the effect on clinical care were infrequently reported. CONCLUSIONS: While there is evidence to suggest that vc is both feasible and effective for use in the clinical care of oncology patients, studies are generally small and methodologically weak, with limited power of inference.

Re-oxygenation improves hypoxia-induced pulp cell arrest.

Dental pulp cells can be exposed to hypoxia during severe inflammation or restorative procedures, though their response to hypoxia is not well-understood. We hypothesized that hypoxia has effects on the growth of pulp cells in vitro. When the cells were exposed to hypoxia for 48 hr, cell growth was suppressed, and cell death was detected by Hoechst staining. Western blot analysis revealed that phosphorylation of retinoblastoma protein was inhibited in cells exposed to hypoxia. Analyses of the molecules involved in retinoblastoma protein phosphorylation revealed that hypoxia suppressed cyclin D2 and activated p21(CIP1/WAF1). Further, hypoxia-exposed pulp cells showed improvement of cell viability, cell-cycle progression, and expression of cyclin D2 with re-oxygenation. These findings indicate that hypoxia-induced cell cycle arrest in pulp cells is reversible, while cyclin D2 may play an essential role in the improvement of cell proliferation with re-oxygenation.

Integrin-supported fast rate intracellular delivery of plasmid DNA by extracellular matrix protein embedded calcium phosphate complexes.

Extracellular matrix (ECM) proteins, such as collagen and fibronectin, play vital roles in development and maintenance of hard tissue (bone or tooth) and are, consequently, thoroughly investigated for construction of biomimetic scaffolds in combination with calcium phosphate (CaP) material (the major component of hard tissue) for bone or dental tissue engineering. Realizing the natural affinity of ECM components toward inorganic constituents of hard tissue, we successfully constructed the nanohybrids of DNA/CaP particles with either collagen 1 or fibronectin, which finally possessed the capability of specific recognition of integrin receptor for being swiftly internalized across the plasma membrane, leading to remarkably high transgene expression in mammalian cells. This new approach with precise receptor-specific delivery as well as 10- to 50-fold enhanced efficiency level compared to the classical one, has immediate applications for basic research and large scale production of recombinant therapeutic proteins and looks promising for gene therapy.

Efficacy of ozone on survival and permeability of oral microorganisms.

In the present study, we examined the effect of ozonated water on oral microorganisms and dental plaque. Almost no microorganisms were detected after being treated with ozonated water (4 mg/l) for 10 s. To estimate the ozonated water-treated Streptococcus mutans, bacterial cells were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Fluorescence microscopic analysis revealed that S. mutans cells were killed instantaneously in ozonated water. Some breakage of ozonated water-treated S. mutans was found by electron microscopy. When the experimental dental plaque was exposed to ozonated water, the number of viable S. mutans remarkably decreased. Ozonated water strongly inhibited the accumulation of experimental dental plaque in vitro. After the dental plaque samples from human subjects were exposed to ozonated water in vitro, almost no viable bacterial cells were detected. These results suggest that ozonated water should be useful in reducing the infections caused by oral microorganisms in dental plaque.

Transient co-localization of c-Jun N-terminal kinase and c-Jun with heat shock protein 70 in pulp cells during apoptosis.

The c-Jun N-terminal kinase (JNK) pathway and heat-shock proteins (HSPs) are involved in stress-induced apoptosis. Here we examined the association of JNK, c-Jun, and anti-apoptotic HSPs with pulp apoptosis during wound healing. In normal pulp, c-Jun was activated only in a few pulp cells, but JNK was not. HSP70 was expressed in the cytoplasm of pulp cells. One day after injury, active JNK and c-Jun were detected in apoptotic pulp cells, whereas HSP70 was detected in non-apoptotic cells. We also found the translocation of HSP70 into nuclei of pulp cells, and co-localization with active JNK and c-Jun. Four days after injury, active JNK and c-Jun disappeared in pulp cells, and HSP70 was relocalized from nuclei to the cytoplasm. These results suggest that the JNK pathway may be one of the compartments inducing apoptosis in pulp cells, and that HSP70 may have an inhibitory role in the apoptosis of pulp cells during wound healing.

Primary and secondary induction of apoptosis in odontoblasts after cavity preparation of rat molars.

The death regulation of damaged pulp cells after cavity preparation is not well-known. In this study, we examined whether apoptosis is associated with the death regulation of damaged pulp cells. In normal rat molars, terminal deoxynucleotidyl transferase-mediated labeling (TUNEL)-positive cells were not observed. Just after surgery, odontoblasts under cavities were TUNEL-positive, and these signals disappeared in six hours. One day after surgery, we found the reappearance of TUNEL-positive cells in the subodontoblastic region under cavities, and positive signals disappeared in four days. Ultrastructure of TUNEL-positive cells showed characteristics typical of apoptotic cells. Phagocytosis of apoptotic cells by scavenger cells was also observed. By immunohistochemistry, we also found Bcl-2-positive odontoblasts one day after surgery. These results suggest that two waves of apoptosis are induced in odontoblasts after cavity preparation, and that apoptotic cells must be eliminated before the initiation of reparative dentinogenesis.

Temporal and spatial expression of c-jun and jun-B proto-oncogenes in pulp cells involved with reparative dentinogenesis after cavity preparation of rat molars.

c-jun and jun-B are nuclear proto-oncogenes induced by growth factors such as bone morphogenetic proteins (BMPs). These gene products enhance the expression of many genes, including osteocalcin and collagen types, indicating that c-jun and jun-B play important roles in the cell differentiation process. It is also known that BMPs affect the differentiation of pulp cells to odontoblast-like cells during reparative dentinogenesis, but little is known about the transcriptional regulation of genes in cells associated with reparative dentinogenesis. In this study, we examined the expression of c-jun and jun-B in pulp cells during reparative dentinogenesis after cavity preparation of rat molars by in situ hybridization. In rat tooth germs, c-jun and jun-B were co-expressed in the odontoblastic lineage. In rat adult molars, c-jun was expressed in the odontoblast layer, but the jun-B expression was absent in all pulp cells. After cavity preparation, we found that c-jun and jun-B were coexpressed in pulp cells underneath cavities. During the early phase of reparative dentinogenesis, levels of c-jun and jun-B greatly increased in pulp cells within and around the reparative dentin matrix formed adjacent to the cavity floor. Fourteen days after cavity preparation, c-jun and jun-B were expressed only in pulp cells lining the irregular surface of the thick reparative dentin. These results suggest that c-jun and jun-B may play important roles both in physiological and in reparative dentinogenesis; in particular, the limited distribution of the jun-B expression suggests a specific role of jun-B only in cells involved with the active formation of the dentin matrix during primary and reparative dentinogenesis.

Expressions of c-jun and jun-B proto-oncogenes in odontoblasts during development of bovine tooth germs.

c-jun and jun-B genes are among the nuclear proto-oncogenes induced by growth factors such as the TGF-beta superfamily and play important roles in cell differentiation. These gene products enhance expressions of proteins including osteocalcin, alkaline phosphatase, and collagens. On the other hand, it is well-known that the TGF-beta superfamily affects odontoblast differentiation, and that differentiated odontoblasts express extracellular and membrane proteins as described above. However, there are few reports of factors that participate in the transcriptional regulation of odontoblasts. Especially, little is known about the expression of c-jun and jun-B genes. In this study, we focused on the examination of expressions of c-jun and jun-B genes in dental papillae of bovine tooth germs. Using in situ hybridization, we found that these genes were expressed only in the odontoblastic lineage, but not in other dental papilla cells. Levels of c-jun and jun-B mRNAs increased along the gradient of differentiation of odontoblasts. These levels of c-jun mRNAs were maintained in both young and mature odontoblasts. However, unlike the c-jun gene, expression of the jun-B gene became sparse in mature odontoblasts compared with young odontoblasts. For further analysis, Northern hybridization of total RNA extracted from differentiated odontoblasts was performed for the examination of levels of jun-B mRNAs, indicating that levels of jun-B mRNAs of mature odontoblasts were clearly less than those of young odontoblasts. These results suggest that c-jun and jun-B genes may participate in the transcriptional regulation of odontoblasts of bovine tooth germs, and may control the odontoblast phenotype. Furthermore, our results suggest that these genes can be markers of odontoblasts during dentinogenesis; especially, high expression of jun-B gene can be a marker of young odontoblasts that start to form the new dentin matrix.

Frequência de hemoglobinopatia S detectada no Instituto Adolfo Lutz Central e sua importãncia em saúde pública / Frequency of hemoglobinopathie S detected in Adolfo Lutz Institute and its importance on Public Health

A incidência de hemoglobia S foi investigada em 1575 amostras de sangue periférico da rotina hematológica do IAL de março a junho de 1994, através da pesquisa de células falciformes nos esfregaços sanguíneas corados por Leishman, pela prova de falcização com metabissulfito de sódio. No esfregaço de sangue periférico foi observada a presença de células falciformes em 21 lâminas (1,33%). A frequência de positividade nos testes de solubilidade e prova de falcização foi de 3,05% (48 casos), sendo todos confirmados por eletroforese de hemoglobina em acetato de celulose pH 8,6 e em gel de ágar citrato pH 6,2, onde observou-se 2,92% de HbAs e 0,06% de HbSC. Devido a grande miscigenação racial da população brasileira o diagnóstico laboratorial da hemoglobinopatia S torna-se de vital importância em termos de Saúde Pública.

Frequency of Candida sp infection in tuberculous patients with acquired immunodeficiency syndrome: morphological and immunocytochemical study in sputum.

Sputum samples from AIDS patients with and without pulmonary tuberculosis were analyzed morphologically and immunocytochemically to determine the frequency of occurrence of Candida sp. Mycobacterial infection was detected by bacterioscopy and/or culture and cytological evaluation was performed using Papanicolaou and Toluidine Blue staining. Immunoreaction for Candida sp was performed using polyclonal antibody in selected cases with fungal structures in smears stained by the Papanicolaou or Toluidine Blue method. An increased frequency of Candida sp (2.5 times) was observed in the tuberculous group compared to the group of AIDS patients without tuberculosis. The Toluidine Blue stain showed good results for the detection of Candida sp in sputum. Due to the increased risk of this opportunistic infection among more severely immunocompromised patients. Toluidine Blue staining of sputum samples submitted to analysis seems to be a reliable screening method.

Hypertrophic motor and sensory neuropathy type I (Charcot-Marie-Tooth disease): ultrastructural study of sural nerve biopsy in members of a family.

Three members of a family with a hereditary neuropathy were studied. Light, electron microscopy and teasing of isolated fibres were performed. The findings confirmed the clinical and electrophysiological hypothesis of hypertrophic form of Charcot-Marie-Tooth disease. Hypertrophy of Schwann cells with the formation of onion bulb figures as the most evident ultrastructural feature, besides demyelination, remyelination and mild axonal degeneration. Recent data about the genetic transmission and pathogenesis of the hereditary motor and sensory neuropathies (HMSN) are discussed.

The presence of vesicles containing lactate dehydrogenase in the ameloblast layer of bovine enamel organ.

Ameloblast layers were removed from bovine enamel organs, followed by the separation of the extracellular matrix-vesicle fraction after collagenase digestion. Lactate dehydrogenase (LDH)-containing vesicles were found in that fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of ameloblasts, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles. The existence of LDH-containing vesicles in enamel organ, where mineralization had been believed not to be initiated by matrix vesicles, suggests the possibility that LDH-containing vesicles have a specific function different from that of matrix vesicles.

Presence of vesicles containing lactate dehydrogenase in the dentin of bovine tooth germs.

The dentin was removed from bovine tooth germs, followed by the separation of the extracellular matrix vesicle fraction after collagenase treatment. Lactate dehydrogenase (LDH)-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of odontoblasts. Other cytosolic enzymes were not detected in LDH-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles.

[Enterovirus 70 in the metropolitan region of São Paulo, Brazil, from 1984 to 1987: infection aspects in endemic and epidemic periods]. / Enterovírus-70 na região metropolitana de São Paulo, Brasil, de 1984 a 1987: aspectos da infecção em períodos epidêmico e endêmico.

Results of investigation about Enterovirus-70 (EV-70) as an etiologic agent of epidemic of acute haemorrhagic conjunctivitis (AHC) and neurological disease in the metropolitan area of São Paulo city are presented. During the first three months of 1984, in an epidemic period of AHC, 3 groups with a total of 291 persons were studied. The group A included 90 individuals affected by AHC; the 99 persons belonging to group B did not acquire the AHC but referred familiar contact with the ill individuals; the group C included 102 persons who denied the AHC or any contact with the illness. Neutralization test in BHK-21 cell culture was used for measurement of antibodies in sera. For the detection of the presence of IgM, indirect immunofluorescence assay was utilized. The presence of IgM antibodies was observed in 56.7%, 33.3% and 20.6% of persons belonging to groups A, B and C, respectively. The 10-29 age group was the most affected in the group A. From april 1984, after the end of epidemic period of AHC, until December 1987, three sporadic cases of AHC and 10 cases with acute neurological disease associated with recent infection by EV-70 were observed. Nine of 10 persons with acute neurological symptoms had paralysis of cranial nerves, all of them recovering without sequelae. The circulation of EV-70 in the population during the endemic period was maintained by either asymptomatic, sporadic cases of AHC or neurological diseases.