Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice.

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti-cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase-3 (MMP-3) and cytokine level in serum by enzyme-linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP-3, interleukin (IL)-2, IL-6, CXCL1 and macrophage inflammatory protein (MIP)-1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.

Interleukin-8 induces DNA synthesis, migration and down-regulation of cleaved caspase-3 in cultured human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC). MATERIAL AND METHODS: DNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit. RESULTS: IL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC. CONCLUSION: IL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells.

Irsogladine maleate inhibits Porphyromonas gingivalis-mediated expression of toll-like receptor 2 and interleukin-8 in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Periodontitis is an infectious disease caused by an interaction between the host and periodontopathogenic bacteria. Regulating the immune response in human gingival epithelial cells (HGEC) may contribute to the prevention of periodontitis. Irsogladine maleate (IM) has previously been shown to regulate inflammation and the cell-cell junctional barrier in HGEC. In addition to these functions, control of bacterial recognition is important for preventing inflammation in periodontal tissue. Innate immunity in gingival epithelium is the first line of defense and plays a crucial role against bacterial challenge. Therefore, the effect of IM on regulating toll-like receptor 2 (TLR2), which is part of the innate immunity, was determined in this study. MATERIAL AND METHODS: OBA-9, an immortalized human gingival epithelial cell line, and primary cultured HGEC were used in this study. Real-time PCR and western blotting were performed in OBA-9 or HGEC stimulated with whole cells of Porphyromonas gingivalis or with lipopolysaccharide (LPS) derived from P. gingivalis (PgLPS) in the presence or absence of IM to determine expression of TLR2 mRNA and production of TLR2 protein. Small interfering RNA (siRNA) against TLR2 was transfected into OBA-9 to clarify the association between the induction of TLR2 and interleukin-8 (IL-8) production. RESULTS: The addition of IM into P. gingivalis or PgLPS-induced OBA-9 suppressed IL-8 production (p < 0.01). The addition of IM also abolished the induction of TLR2 by P. gingivalis or PgLPS in OBA-9 and primary cultured HGEC (p < 0.01). The suppressive effect of IM on the induction of TLR2 was also confirmed by immunohistostaining. Stimulation with peptidoglycan, a specific ligand for TLR2, suppressed the expression of toll-like receptor 4 (TLR4) mRNA in the presence of IM (p < 0.01). However, LPS derived from Escherichia coli, a ligand for TLR4, did not induce the expression of TLR2 mRNA. The PgLPS-induced expression of TLR4 mRNA was abolished by IM. Knockdown of TLR2 by siRNA transfection resulted in a weaker response of induction of IL8 mRNA in P. gingivalis or PgLPS-stimulated OBA-9. CONCLUSION: These results suggest that IM suppresses the induction of IL-8 production by regulating increased levels of TLR2.

LL37 induces VEGF expression in dental pulp cells through ERK signalling.

AIM: To examine the in vitro effects of LL37 on the expression of vascular endothelial growth factor (VEGF) in human pulp cells and to identify the intracellular signalling pathway involved. METHODOLOGY: Pulp cells at passage 6 were treated with 10 µg mL(-1) synthesized LL37, and an inhibition assay was performed with MAPK or NF-κB inhibitors to determine the possible signalling pathway. VEGF mRNA, VEGF protein and phosphorylated ERK1/2 levels were determined by real-time PCR, ELISA and Western blot, respectively. Data were analysed using t-tests. RESULTS: LL37 significantly increased both the mRNA and protein levels of VEGF in pulp cells (P < 0.01). However, pre-treatment with an ERK kinase inhibitor suppressed these increases. Furthermore, the inhibitor blocked LL37-induced ERK1/2 phosphorylation. CONCLUSIONS: LL37 activated the ERK pathway to boost VEGF secretion from human pulp cells. Because of this angiogenic effect and its reported induction of pulp cell migration and antimicrobial activity against cariogenic bacteria, LL37 may be applicable as a pulp capping material.

Antimicrobial peptide LL37 promotes vascular endothelial growth factor-A expression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: LL37, originally found in the innate immune system, is a robust antimicrobial peptide. LL37 exhibits multiple bio-functions in various cell types, such as migration, cytokine production, apoptosis, and angiogenesis besides its antimicrobial activity Periodontal ligament (PL) cells play a pivotal role in periodontal tissue regeneration. Based on these findings, we hypothesized that LL37 can regulate PL cell function to promote regeneration of periodontal tissue. To prove this hypothesis, we investigated the effect of LL37 on the potent angiogenic inducer vascular endothelial growth factor (VEGF) expression in cultures of human PL (HPL) cells because neovascularization is indispensable for the progress of tissue regeneration. Moreover, we investigated the signaling cascade associated with LL37-induced VEGF expression. MATERIAL AND METHOD: HPL cells were treated with synthesized LL37 in the presence or absence of PD98059, a MEK-ERK inhibitor, or PDTC, an NF-κB inhibitor. VEGF expression levels were assessed by real-time polymerase chain reaction analysis and an enzyme-linked immunoassay. Phosphorylation levels of ERK1/2 or NF-κB p65 were determined by Western blotting. RESULTS: LL37 upregulated VEGF-A expression at the mRNA and protein levels in HPL cells, while VEGF-B mRNA expression was not affected. Both ERK and NF-κB inhibitors clearly abrogated the increase in VEGF-A levels induced by LL37 in HPL cells. Importantly, LL37 increased phosphorylated levels of ERK1/2 and NF-κB p65 in HPL cells. CONCLUSION: LL37 induces VEGF-A production in HPL cells via ERK and NF-κB signaling cascades, which may result in angiogenesis, thereby contributing to periodontal regeneration.

Loss of claudin-1 in lipopolysaccharide-treated periodontal epithelium.

BACKGROUND AND OBJECTIVE: The epithelial barrier is a critical component of innate immunity and provides protection against microbial invasion. Claudin-1, a tight junction protein, is known to contribute to the epithelial cell barrier. An experimentally induced rat periodontal disease model was used to study the effects of lipopolysaccharide (LPS) on the expression of tight junction-associated molecule genes in the junctional epithelium. MATERIAL AND METHODS: LPS was applied for 8 wk in the gingival sulcus, and junctional epithelium was collected by laser-capture microdissection and subjected to microarray analysis. RESULTS: Microarray analysis identified that expression of the claudin-1 gene was decreased in the epithelium by chronic LPS challenge. Immunohistochemical analysis confirmed the expression of claudin-1 protein in junctional epithelium and that 8 wk of chronic LPS topical application significantly reduced claudin-1 expression. The effect of LPS on claudin-1 protein expression was validated using a porcine junctional epithelial cell culture Transwell model. The epithelial barrier, as measured using transmembrane resistance, was significantly reduced after 3 wk of LPS challenge and this was associated with a decreased level of expression of claudin-1 protein. CONCLUSION: These results confirm that the initiation of experimental periodontal disease is associated with reduction in the expression of claudin-1 gene and protein. This decreased level of a critical tight junction protein may result in the disruption of barrier function and may play an important role in the initiation of periodontal disease.

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy.

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.

Chronic nicotine treatment enhances focal ischemic brain injury and depletes free pool of brain microvascular tissue plasminogen activator in rats.

Effects of nicotine treatment (4.5 mg/kg of nicotine-free base/day administered s.c. by osmotic minipumps for 14 days) on focal ischemic stroke and expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in cerebral microvessels were studied in rats in vivo using a reversible (1 h) middle cerebral artery occlusion model. Plasma levels of nicotine and its major metabolite cotinine after 14 days of treatment were 88 and 364 ng/ml, respectively. Nicotine treatment resulted in 35-40% (p < 0.001) decrease in the blood flow in the periphery of the ischemic core during reperfusion, an increase in the neurologic score of 2.6-fold (p < 0.01), and 36% (p < 0.05) and 121% (p < 0.01) increases in the injury and edema volume in the pallium, respectively. A free pool of brain microvascular t-PA antigen was completely depleted by nicotine, while the expression of the PAI-1 antigen and/or PAI-1-t-PA complexes remained unchanged. The relative abundance of cerebromicrovascular t-PA mRNA transcript versus beta-actin mRNA transcript did not change with nicotine. It is concluded that chronic nicotine treatment impairs the restoration of blood flow, worsens the neurologic outcome, and enhances brain injury following an ischemic insult. These nicotine effects are associated with depletion of brain microvascular t-PA antigen.

Results of early surgical evacuation of packed intraventricular hemorrhage from aneurysm rupture in patients with poor-grade subarachnoid hemorrhage.

OBJECT: The goal of this study was to evaluate the results of early surgical evacuation of "packed" intraventricular hemorrhage (IVH) in patients with poor-grade subarachnoid hemorrhage (SAH). METHODS: The authors performed surgery within 24 hours after onset of SAH, identified on neuroimaging as a cast distending the ventricular system, in 74 patients with poor-grade SAH (World Federation of Neurosurgical Societies Grades IV and V) without intracerebral hemorrhage. Eighteen of these patients had packed IVH; in these patients the intraventricular clots were extensively evacuated via frontal corticotomy performed under microscopic view. CONCLUSIONS: Overall, 42% of the 74 patients undergoing craniotomy in the acute stage had favorable outcomes, whereas 30% died. Using multivariate analysis, variables significantly associated with favorable outcome in patients with poor-grade SAH included absence of a packed intraventricular clot on computerized tomography scanning; absence of a history of cardiac disease; and a Glasgow Coma Scale score of 11 or 12. None of the 18 patients who had packed IVH had favorable outcomes and seven of these died. In six recently treated patients with packed IVH, which was examined using fluid-attenuated inversion recovery imaging, extensive periventricular brain damage was found both immediately after surgery and during the chronic stage. Accordingly, the authors believe that irreversible periventricular brain damage is already complete immediately after packed IVH occurs.

Attenuation of brain injury and reduction of neuron-specific enolase by nicardipine in systemic circulation following focal ischemia and reperfusion in a rat model.

A reversible middle cerebral artery occlusion was performed in rats to determine whether nicardipine, a dihydropyridine voltage-sensitive Ca++ channel (VSCC) antagonist, exerts neuroprotective effects when administered 10 minutes following an ischemic insult, and if it does, whether this is due to its vasodilatory action and effect on cerebral blood flow (CBF) or to direct blockade of Ca++ entry into ischemic brain cells. An increase in the intracellular calcium, [Ca++]i, plays a major role in neuronal injury during cerebral ischemia. Although a large amount of Ca++ enters neurons through the VSCC during ischemia, inconsistent neuroprotective effects have been reported with the antagonists of the VSCC. An intraperitoneal injection of nicardipine (1.2 mg/kg) was administered to rats 10 minutes after the onset of ischemia, and 8, 16, and 24 hours after occlusion. Cortical CBF was determined by laser-Doppler flowmetry. Neurological and neuropathological examinations were performed after 72 hours. Neuron-specific enolase, a specific marker for the incidence of neuronal injury, was measured in plasma. The CBF and other physiological parameters were not affected by nicardipine during occlusion or reperfusion. However, nicardipine treatment significantly improved motor neurological outcome by 29%, and the infarction and edema volume in the pallium as well as the edema volume in the striatum were significantly reduced by 27%, 37%, and 52%, respectively. Nicardipine also reduced the neuron-specific enolase plasma levels by 50%, 42%, and 59% at 24, 48, and 72 hours after the occlusion, respectively. It is concluded that nicardipine may attenuate focal ischemic brain injury by exerting direct neuroprotective and antiedematous effects that do not depend on CBF.

Hydrocephalus and epilepsy.

Since the introduction of ventriculo-atrial and/or ventriculo-peritoneal shunting for hydrocephalic patients, controversies have developed regarding the likelihood of epileptic seizures developing as a result of the shunting itself and/or its complications. On the other hand, hydrocephalus is not commonly recognized as a cause of seizures in general, although epilepsy is reported to be frequently associated with shunt-treated hydrocephalus, especially in children. Several authors have reported an increased risk of epileptic seizures after shunt placement, but the underlying mechanisms are still controversial. The insult to the brain at the time of ventricular catheter insertion, the presence of the shunt tube itself as a foreign body, the burr hole location, the number of shunt revisions after malfunction, associated infection, the etiology of hydrocephalus, and associated mental retardation are thought to be related to the risk of epilepsy. Age at the time of initial shunt placement also seems to be an important factor. Early shunting is a well-known determinant of risk in shunt obstruction, and children less than 2 years old are consequently at a higher risk of developing epilepsy than older ones. It is reported that antiepileptic drug treatment is not so reliable as might be expected. Conscientious and more sophisticated EEG recording in those children may be beneficial during follow-up. The incidence of seizures in shunted children is reported to be quite high, ranging from 20% to approximately 50%, so that neurosurgeons should pay more attention to the issue of epilepsy in hydrocephalic children. Although ventriculo-extracranial shunts have been the standard treatment for hydrocephalus for decades, the long-term morbidity, including postshunt epileptic seizures, has to be taken seriously. The use of neuroendoscopic techniques when indicated may ameliorate this problem a great deal in the future.

Brain capillary tissue plasminogen activator in a diabetes stroke model.

BACKGROUND AND PURPOSE: Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury. METHODS: Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion. RESULTS: All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively. CONCLUSIONS: Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.

Thrombomodulin expression in bovine brain capillaries. Anticoagulant function of the blood-brain barrier, regional differences, and regulatory mechanisms.

Thrombomodulin (TM), a key cofactor of the TM-protein C pathway, is of major biologic significance for the antithrombotic properties of endothelial cells. Yet, there is uncertainty whether TM is expressed in brain and what mechanisms govern brain endothelial anticoagulant activity. In this study, bovine brain capillaries were used as an in vitro model of the blood-brain barrier to determine factors involved in the regulation of TM expression in cerebral vasculature. Quantitative competitive-polymerase chain reaction assay revealed significant regional differences in the amount of brain capillary TM mRNA, i.e., cortical > cerebellar > pontine, consistent with the reverse transcription-polymerase chain reaction findings in which the abundance of TM mRNA was analyzed relative to beta-actin mRNA. Regional differences in TM mRNA brain capillary level correlated well with differences in protein C activation. The TM mRNA and activity were not detectable in brain parenchyma. Pathogenic mediators of ischemic stroke, interleukin 1 beta (10 U/mL), and tumor necrosis factor alpha (10 U/mL), produced a time-dependent decrease in brain capillary TM mRNA (t1/2 of 2.1 and 3.9 hours, respectively) and reduced endothelial TM activity. Incubation of brain capillaries with retinoic acid (10 mumol/L) and dibutyryl cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and 8 hours, respectively, followed by an increase in protein C activation. We conclude that TM at the blood-brain barrier is likely to be an important physiologic anticoagulant in brain microcirculation. Its downregulation by cytokines may contribute to ischemic brain damage and potentially could be counteracted by retinoic acid and cAMP.

Effects of chronic metabolic alkalosis on Ca2+, PTH and 1,25(OH)2D3 in the rat.

The effect of chronic metabolic alkalosis on arterial blood ionized calcium concentration ([Ca2+]) and the levels of serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is difficult to predict. Although a fall in pH directly decreases [Ca2+], chronic alkalosis reduces urine calcium excretion, which could elevate [Ca2+]. [Ca2+] modulates the serum level of PTH and the level of 1,25(OH)2D3 directly and through PTH. To determine the effect of chronic metabolic alkalosis on [Ca2+], PTH, and 1,25(OH)2D3, rats were made alkalemic by feeding a chloride-deficient diet (LCl) or LCl with 75 mM NaHCO3 in the drinking water (LCl + HCO3-) and compared with controls fed a chloride-replete diet (NCl). Compared with NCl, after 8 days of LCl and LCl + HCO3- arterial pH and PTH rose and [Ca2+] fell. Serum 1,25(OH)2D3 tended to rise with LCl and rose with LCl + HCO3-. Serum 1,25(OH)2D3 was correlated inversely with [Ca2+] (r = -0.510, n = 54, P less than 0.001) and with pH (r = -0.291, n = 57, P less than 0.03) but not with PTH or phosphorus. Stepwise regression analysis indicated that [Ca2+] accounted for the majority of the variance of serum 1,25(OH)2D3. Chronic metabolic alkalosis induced by a low-chloride diet and HCO3- appears to increase serum PTH and 1,25(OH)2D3 through a fall in [Ca2+].