Epstein-Barr virus reactivation by persistent apical periodontal pathogens.

AIM: To assess whether Epstein-Barr virus (EBV) reactivation is triggered by persistent apical periodontitis-related microbes using in vitro and ex vivo methodologies. METHODOLOGY: Surgically removed human periapical granulomas (n = 50) and healthy gingival tissues (n = 10) were analysed to determine the presence of EBV and seven persistent apical periodontitis-related microbes. In addition, real-time polymerase chain reaction was used to detect the mRNA expression of BZLF-1, an immediate-early gene of EBV. Expression of latent membrane protein (LMP)-1 and ZEBRA, an early lytic protein of EBV encoded by BZLF-1, was also examined using triple-colour immunofluorescence staining. n-Butyric acid produced by the microbes was quantified, and luciferase assays were performed in association with bacterial lysates. In addition, Daudi cells were cultured with bacterial lysates, and the expression levels of BZLF-1 mRNA and ZEBRA protein were determined. RESULTS: EBV DNA and BZLF-1 mRNA were detected in 47 out of 50 periapical granulomas, but not in healthy gingival tissues. The EBV DNA copy number and the number of Fusobacterium nucleatum were significantly positively correlated with BZLF-1 expression in periapical granulomas. The number of Prevotella intermedia was slightly correlated with BZLF-1 expression; however, the other microbes were not. CD79a-positive B cells in periapical granulomas, but not those in healthy gingival tissues, expressed both LMP-1 and ZEBRA. n-Butyric acid production was the highest in F. nucleatum and the lowest in P. intermedia. Enterococcus faecalis, Candida albicans and the other tested microbes did not produce n-butyric acid. An F. nucleatum lysate exhibited significantly increased BZLF-1-luciferase activity in the same manner of commercial butyric acid, whereas P. intermedia did not. F. nucleatum also induced the expression of BZLF-1 mRNA and ZEBRA protein by Daudi cells, indicating that EBV reactivation was induced. CONCLUSION: Among the persistent apical periodontitis-related bacteria that were tested, F. nucleatum most strongly reactivated latent EBV, whereas E. faecalis and C. albicans as well as the other microbes did not.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

AIM: To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). METHODOLOGY: The concentrations of short-chain fatty acids (SCFAs) in P. endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P. endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P. endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P. endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. RESULTS: The concentrations of n-butyric acid in P. endodontalis culture supernatants were significantly higher than those of other SCFAs (P = 0.0173). Using B-95-8-221 Luc cells treated with P. endodontalis culture supernatants, the luciferase assay demonstrated that P. endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P. endodontalis culture supernatants. P. endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P. endodontalis cells in each specimen. CONCLUSIONS: n-butyric acid produced by P. endodontalis reactivated latent EBV.

A potential role for the silent information regulator 2 homologue 1 (SIRT1) in periapical periodontitis.

AIM: To investigate the role played by silent information regulator 2 homologue 1 (SIRT1) during angiogenesis of periapical periodontitis. METHODOLOGY: Periapical granulomas were subjected to dual-colour immunofluorescence imaging and real-time polymerase chain reactions assaying the expression levels of SIRT1, vascular endothelial growth factor (VEGF) and VE-cadherin. The association between Ki-67 and SIRT1 expression was also examined. Human umbilical vein endothelial cells (HUVECs) were treated with a combination of lipopolysaccharide and resveratrol (a SIRT1 activator) or sirtinol (a SIRT1 inhibitor); and the levels of mRNAs encoding SIRT1, VEGF and VE-cadherin were determined. HUVEC tube formation was assayed in the presence of resveratrol or sirtinol. The Mann-Whitney U-test or the Tukey-Kramer test was used for statistical analysis. RESULTS: Ki-67-expressing cells, including endothelial cells, lay adjacent to SIRT1-expressing cells in periapical granulomas. In addition, SIRT1-expressing cells were detected adjacent to VEGF-expressing cells and VEGF- or VE-cadherin-expressing endothelial cells. SIRT1, VEGF and VE-cadherin mRNA expression levels in periapical granulomas were significantly higher (P = 0.0054, 0.0090 and 0.0090, respectively) than those in healthy gingival tissues. HUVECs treated with resveratrol exhibited significantly higher expression of mRNAs encoding SIRT1, VEGF and VE-cadherin (P = 0.0019, 0.00005 and 0.0045, respectively) compared with controls, but sirtinol inhibited such expression. Resveratrol caused HUVECs to form tube-like structures, whilst sirtinol inhibited this process. CONCLUSIONS: These findings suggest that SIRT1 may stimulate angiogenesis in periapical granulomas by triggering the proliferation of endothelial cells and inducing VEGF and VE-cadherin expression.

Generation and characterization of immortalized human microglial cell lines: expression of cytokines and chemokines.

Microglia are a major glial component of the central nervous system (CNS), play a critical role as resident immunocompetent and phagocytic cells in the CNS, and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS. Studies of human microglia have been hampered by the difficulty of obtaining sufficient numbers of human microglia. One way to circumvent this difficulty is to establish permanent cell lines of human microglia. In the present study we report the generation of immortalized human microglial cell line, HMO6, from human embryonic telencephalon tissue using a retroviral vector encoding myc oncogene. The HMO6 cells exhibited cell type-specific antigens for microglia-macrophage lineage cells including CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and ricinus communis agglutinin lectin-1 (RCA), and actively phagocytosed latex beads. In addition, HMO6 cells showed ATP-induced responses similar to human primary microglia in Ca2+ influx spectroscopy. Both human primary microglia and HMO6 cells showed the similar cytokine gene expression in IL-1beta, IL-6, IL-8, IL-10, IL-12, IL-15, and TNF-alpha. Using HMO6 cells, we investigated whether activation was induced by Amyloid-beta fragments or lipopolysaccharide (LPS). Treatment of HMO6 cells with Amyloid-beta 25-35 fragment (Abeta(25-35)) or Amyloid-beta 1-42 fragment (Abeta(1-42)) led to increased expression of mRNA levels of cytokine/chemokine IL-8, IL-10, IL-12, MIP-1beta MIP-1, and MCP-1, and treatment with LPS produced same results. Expression of TNF-alpha and MIP1-alpha was not detected in unstimulated HMO6 cells, but their expression was later induced by long-term exposure to Abeta(25-35) or Abeta(1-42.) ELISA assays of spent culture media showed increased protein levels of TNF-alpha and IL-8 in HMO6 cells following treatment with Abeta(25-35) or LPS. Taken together, our results demonstrate that treatment of human primary microglia and HMO6 immortalized human microglia cell line with Abeta(25-35), Abeta(1-42) and LPS upregulate gene expression and protein production of proinflammatory cytokines and chemokines in these cells. The human microglial cell line HMO6 exhibits similar properties to those documented in human microglia and should have considerable utility as an in vitro model for the studies of human microglia in health and disease.

Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture.

Erythropoietin (EPO) is a hematopoietic growth factor that stimulates proliferation and differentiation of erythroid precursor cells and is also known to exert neurotrophic activity in the central nervous system (CNS). However, little is known about expression of EPO and EPO receptor (EPOR) in human CNS tissues. In the present study, we investigated the effects of proinflammatory cytokines on EPO and EPOR expression in highly purified cultures of human neurons, astrocytes, microglia, and oligodendrocytes using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). EPO mRNA was demonstrated only in human astrocytes, while EPOR expression was found in human neurons, astrocytes, and microglia. Neither EPO nor EPOR expression was found in oligodendrocytes. In human astrocytes, EPO mRNA and secreted EPO protein levels were downregulated after exposure to proinflammatory cytokines (IL-1beta, IL-6, or TNF-alpha). In human neurons, TNF-alpha treatment markedly increased EPOR expression. These results suggest that proinflammatory cytokines regulate expression of EPO and EPOR in human neurons, astrocytes, and microglia and further facilitate interactions among different cell types in the human CNS.

Synthesizing oligomers from monomeric nucleotides in simulated hydrothermal environments.

Dimers and trimers of adenosine monophosphate (AMP) were synthesized from AMP in environments simulating hot vents on the sea floor of the primitive Earth. The simulated environments were made in the flow reactor, in which an aqueous solution of reactants was circulated from the hot to the cold region repeatedly. The oligomerization proceeded most significantly when the hot reaction solution at about 110 degrees C was abruptly ejected into the cold environment maintained at about 0 degree C.

Onset of the sliding movement of an actin filament on myosin molecules: from isotropic to anisotropic fluctuations.

An actin filament contacting myosin molecules increased the fluctuation intensity of the filamental displacement as the ATP concentration increased. In particular, fluctuations in the filamental displacement in the planar plane in which the sliding movement takes place were isotropic at a low ATP concentration, and became anisotropic as the concentration increased. The build-up of the sliding movement of an actin filament was associated with the transformation from isotropic to anisotropic fluctuations of the filamental displacement.

Contractile and protractile coordination within an actin filament sliding on myosin molecules.

An actin filament exhibits distortions longitudinally when it slides upon myosin molecules. We observed that the actin filament demonstrated contractile distortions at low ATP concentrations and protractile distortions at high concentrations. Temporal development of such distortions was identified, by tracing each of several speckled fluorescent markers attached to the actin filament. Close association of the sliding movement to the moving distortions of an actin filament suggests the presence of a unitary mechanism regulating the apparently two different modes of dynamic movement.

Staggered movement of an actin filament sliding on myosin molecules in the presence of ATP.

An actin filament sliding on myosin molecules in the presence of an extremely low concentration of ATP exhibited a staggered movement. Longitudinally sliding movement of the filament was frequently interrupted by its non-sliding, fluctuating movements both in the longitudinal and transversal directions. Intermittent sliding movements of an actin filament indicate establishment of a coordination of ATP-mediated active sites distributed along the filament.

Regulated crosslinked actin filaments and the decoupling between their ATPase activity and sliding motility.

Troponin-tropomyosin complex from skeletal muscles was observed to regulate sliding movement of actin filaments on myosin molecules in a manner independent of their ATPase activity. When actin molecules were crosslinked with DSS (disuccinimidyl suberate), the myosin ATPase activity in the presence of the modified actin filaments complexed with both troponin and tropomyosin was only 10% less than that in the case of unmodified actin, and the ATPase activation was independent of calcium ions. In contrast, the sliding velocity of the modified actin filaments on myosin molecules decreased to zero below pCa 6.5. The present results indicate that troponin-tropomyosin complex regulates contractile movement of actomyosin systems through direct alternation of a mechanochemical property of the thin filaments, not through a decrease in the ATPase activity of the myosin molecules.

Communicative interaction of myosins along an actin filament in the presence of ATP.

Myosin molecules contacting an actin filament in the presence of ATP were found to regulate the filamental fluctuations due to ATP hydrolysis in a communicative manner along the filament. As an evidence of the occurrence of the communication, ATP-activated fluctuating displacements of the filament in the direction perpendicular to its longitudinal axis were identified to propagate at a finite velocity not less than about 0.2 micron/s unidirectionally along the filament.

Dual role of tropomyosin on chemically modified actin filaments from skeletal muscle.

Actin filaments copolymerized with both intact and chemically modified actin monomers restored their sliding activity when they were supplemented with tropomyosin extracted from skeletal muscle. In contrast, the ATPase activation of the copolymers was decreased when supplemented with tropomyosin. The results indicate that tropomyosin along with actin monomers may facilitate sliding activity of the entire actin filament but suppress ATPase activation of intact actin monomers themselves. Accordingly, tropomyosin molecules could be viewed as playing a dual role of both mechanical and chemical regulation of actin monomers.

ATP-dependent fluctuations of single actin filaments in vitro.

Single filaments of actin were observed to fluctuate in the direction perpendicular to their longitudinal axes when they hydrolyzed ATP in the presence of myosin. The transversal fluctuations of actin filaments were identified by reading the transversal displacements of the filaments under a fluorescence microscope. The transversal fluctuations in the absence of ATP decreased their intensity as the number of myosin molecules contacting directly with actin filaments increased. In the presence of ATP, on the other hand, the amplitude of the transversal fluctuations increased in proportion to the ATP concentration up to a certain level, while the sliding velocity of the filaments did not increase significantly over the same range of ATP concentration. The present observation suggests that the chemical energy released from ATP along actin filament binding to myosin molecules is first and primarily converted into kinetic energy of fluctuations in the form of the displacement movements of the filaments in the direction perpendicular to their longitudinal axes.

Decorating actin filaments with troponin T-I complexes and acceleration of their sliding movement on myosin molecules.

Actin filaments, when partially decorated with troponin T-I complexes, can slide faster on myosin heads than those with no decoration. Purified troponin T-I complexes bind to actins, and inhibit the actin activated myosin adenosine 5'-triphosphatase activity completely when the molar ratio of troponin T-I complex to actin is increased to 1 to 1. Those actin filaments decorated with troponin T-I complexes up to 20 to 50% of their molar ratio exhibit enhancement of the velocity of sliding on myosins up to 20% compared to those without such decoration. As the molar ratio of decoration further increases, the sliding velocity decreases. These results are consistent with the observation that even if some of actin monomers do not participate in the ATPase activity directly, they can interact with myosin heads and take part in the sliding movement.

ATP hydrolysis and sliding movement of actomyosin complex in the presence of ethanol.

We measured both the ATPase activity of actomyosin complex and the sliding velocity of actin filaments on myosin heads under hydrophobic conditions in the presence of ethanol. Both the ATPase activity and the sliding velocity decrease with the increase of ethanol concentration, if the ionic strength is not too high. As ionic strength increases, there appears an optimum concentration of ethanol that can enhance both the ATPase activity and the sliding velocity.

[A 37-year-old man with memory loss, homonymous hemianopsia, and elevation of anti-herpes simplex virus antibody titer].

We report a 37-year-old man who presented memory loss, homonymous right hemianopsia, and elevation of anti-herpes simplex antibody titer. He had an auto accident in January 1992 in that the car he was driving slipped down a 3 m slope; his car was severely damaged, however, he himself was not injured. Shortly after this accident, he went out of his house less often than before, and he noted some difficulty in his vision. He changed his glasses twice, but his vision was unchanged. In July of 1992, he had an onset of difficulty in recent memory and disorientation to time. He also noted diplopia, and difficulty in seeing objects in his right visual field. He was admitted to our hospital on August 26 of the same year. General physical examination was unremarkable. On neurologic examination, he was alert but disoriented to time and place; calculation was also impaired. Mini-mental state examination was 18/30. He had no aphasia, apraxia, or agnosia. He showed a tendency to neglect his left side. Optic fundi and visual acuity were normal; right homonymous hemianopsia was present. Ocular movement was moderately restricted to most of the directions; pupils were isocoric and reacted to light promptly. He complained of diplopia in right gaze, and monocular nystagmus was induced in his right eye upon right lateral gaze. Trigeminal nerves appeared intact. Minimum left facial weakness was present. The remaining of the cranial nerves appeared intact. His gait was wide-based and tandem gait was impossible. Muscle strength was normal as was the muscle tone. Finger to nose and heel to knee tests were done normally.(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of brainstem vascular malformation with isolated trochlear nerve palsy as the initial symptom].

We report a 46-year-old, non-hypertensive man who suddenly developed isolated right trochlear nerve palsy. His diplopia was most prominent in the left lower gaze, and partially alleviated by head tilt to the left or by anteflexion of the neck. His CT scans showed a small high density area consistent with a hemorrhage in the lateral side of the right mesencephalic tectum. His MRI (T2-weighted images) showed a lesion consisting of mixed high- and iso-intensity areas with linear low intensity areas. The margin of the lesion was irregular and nodular. Cerebral angiography (prolonged injection) showed small feeding arteries (or capillaries) in the late arterial phase and dilated draining veins in the venous phase. No tumor stain, early draining veins, or capillary brushes were present. We thought he had an angioma (vascular malformation). AVM seemed unlikely. Review of the literature revealed that trochlear nerve palsy caused by a mesencephalic angioma is extremely rare. MRI and cerebral angiography (prolonged injection) seemed useful for the diagnosis of angiomas (Vascular malformations).