Genetic variation in the recognition of Porphyromonas gingivalis antigens in mice

T-cell cytokine profiles, anti Porphyromonas gingivalis antibodies and Western blot analysis of antibody responses were examined in BALB/c, CBA/CaH, C57BL6 and DBA/2J mice immunized intraperitoneally with different doses of P. gingivalis outer membrane antigens, Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-LD by FAGS analysis and levels of anti-P. gingivalis antibodies in the serum samples determined by enzyme-linked immunosorbent assay. Western blot analysis was performed on the sera from mice immunized with 100 mug of P. gingivalis antigens. The four strains of mice demonstrated varying degrees of T-cell immunity although the T-cell cytokine profiles exhibited by each strain were not affected by different immunizing doses. While BALB/c and DBA/2J mice exhibited responses that peaked at immunizing doses of 100-200 mug of P. gingivalis antigens, CBA/CaH and C57BL6 demonstrated weak T-cell responsiveness compared with control mice. Like the T-cell responses, serum antibody levels were not dose dependent. DBA/23 exhibited the lowest levels of anti-P. gingivalis antibodies followed by BALB/c with CBA/CaH and C57BL6 mice demonstrating the highest levels. Western blot analysis showed that there were differences in reactivity between the strains to a group of 13 antigens ranging in molecular weight from 15 to 43 kDa. Antibody responses to a number of these bands in BALB/c mice were of low density, whereas CBA/CaH and C57BL6 mice demonstrated high-density bands and DBA/2J mice showed medium to high responses. In conclusion, different immunizing doses of P. gingivalis outer membrane antigens had little effect on the T-cell cytokine responses and serum anti-P. gingivalis antibody levels. Western blot analysis, however, indicated that the four strains of mice exhibited different reactivity to some lower-molecular-weight antigens. Future studies are required to determine the significance of these differences, which may affect the outcome of P. gingivalis infection.

Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells

Background: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. Methods: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. Results: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. Conclusions: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis: of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Influence of Antiplatelet Drugs in the Pathogenesis of Experimental Periodontitis and Periodontal Repair in Rats

Background: Platelets contain an array of biologic mediators that can modulate inflammation and repair processes including proinflammatory mediators and growth factors. Previous studies have shown that periodontitis and periodontal repair are associated with platelet activation. We hypothesized that drug-induced platelet inactivation may interfere in the processes of inflammation and repair in experimental periodontitis in rats by suppressing the release of biologic mediators from platelets to the site of injury. Methods: To measure the effects on periodontitis, ligatures were placed around first molars, and aspirin (Asp, 30 mg/kg) or clopidogrel (Clo, 75 mg/kg) was given intragastrically once daily for 15 days. Interleukin-6 (IL-6), tumor necrosis factor-a (TNF-alpha), and thromboxane A(2) levels were measured by enzyme-linked immunosorbent assay. To evaluate the effects of antiplatelet drugs on periodontal repair, ligatures were removed after 15 days of periodontitis induction, and Asp or Clo were administered beginning the following day for 15 days. Periodontal repair was assessed by microcomputed tomography. Results: On periodontitis phase, Asp and Clo significantly reduced levels of TNF-alpha and II-6 (P < 0.05), but only Asp decreased thromboxane A(2) (P < 0.05). Asp and Clo decreased inflammatory infiltration; however, this reduction was more pronounced with Clo treatment (P < 0.05). Histometric analysis showed that Asp and Clo impaired alveolar bone resorption. During the repair phase and after removal of the ligatures, microcomputed tomography analysis demonstrated that treatment with Asp and Clo did not impair alveolar bone repair. Conclusion: Systemic administration of Asp and Clo attenuates the inflammation associated with periodontitis without affecting the repair process when stimulus is removed. J Periodontol 2011;82:767-777.

The Relationship between Blood and Serum Lead Levels in Peripartum Women and their Respective Umbilical Cords

Foetal exposure to lead (Pb) during pregnancy is a major problem. However, no previous study has examined whether Pb concentrations in blood (Pb-B) and in serum (Pb-S) from pregnant women correlate with Pb-B and Pb-S in the foetuses. This hypothesis was tested in the present study. We measured Pb-B and Pb-S in 120 healthy pregnant women (more than 38 weeks of gestation) and their respective umbilical cord samples. The analyses were carried out with an inductively coupled plasma mass spectrometer. We found higher Pb-B levels in the women compared with their respective umbilical cord samples (1.736 +/- 0.090 mu g/dL and 1.194 +/- 0.062 mu g/dL, respectively; p < 0.05). In parallel, we found higher Pb-S levels in the women compared with their respective umbilical cord samples (0.042 +/- 0.003 mu g/dL and 0.032 +/- 0.003 mu g/dL, respectively; p < 0.05). However, similar %Pb-S/Pb-B ratios were found in the women compared with their respective umbilical cord samples (2.414 +/- 0.210% and 2.740 +/- 0.219%, respectively; p > 0.05). Interestingly, we found positive correlations between Pb-B in the umbilical cords and Pb-B in the respective pregnant women (rs = 0.5714; p < 0.0001), and between Pb-S in the umbilical cords and Pb-S in the respective pregnant women (rs = 0.3902; p < 0.0001) as well as between %Pb-B/Pb-S in the umbilical cords and %Pb-B/Pb-S in the respective pregnant women (rs = 0.3767; p < 0.0001). These results indicate that the assessment of Pb-B and Pb-S in pregnant women provides relevant indexes of foetal exposure to Pb. Moreover, the similar %Pb-S/Pb-B in pregnant women and in the umbilical cords shows that the foetuses are directly exposed to the rapidly exchangeable Pb fraction found in their mothers.

Vitamin D receptor haplotypes affect lead levels during pregnancy

Pregnant women are particularly susceptible to toxic effects associated with lead (Pb) exposure. Pb accumulates in bone tissue and is rapidly mobilized from bones during pregnancy, thus resulting in fetal contamination. While vitamin D receptor (VDR) polymorphisms modify bone mineralization and affect Pb biomarkers including blood (Pb-B) and serum (Pb-S) Pb concentrations, and %Pb-S/Pb-B ratio, the effects of these polymorphisms on Pb levels in pregnant women are unknown. This study aimed at examining the effects of three (Fokl, Bsml and Apal) VDR polymorphisms (and VDR haplotypes) on Pb levels in pregnant women. Pb-B and Pb-S were determined by inductively coupled plasma mass spectrometry in samples from 256 healthy pregnant women and their respective umbilical cords. Genotypes for the VDR polymorphisms were determined by PCR and restriction fragment length digestion. While the three VDR polymorphisms had no significant effects on Pb-B, Pb-S or %Pb-S/Pb-B ratio, the haplotype combining the f, a, and b alleles for the Fokl, Apal and Bsml polymorphisms, respectively, was associated with significantly lower Pb-S and %Pb-S/Pb-B (P<0.05). However, maternal VDR haplotypes had no effects on Pb levels in the umbilical cords. To our knowledge, this is the first study showing that a combination of genetic polymorphisms (haplotype) commonly found in the VDR gene affects Pb-S and %Pb-S/Pb-B ratios in pregnant women. These findings may have major implications for Pb toxicity because they may help to predict the existence of a group of subjects that is genetically less prone to Pb toxicity during pregnancy. (C) 2010 Elsevier B.V. All rights reserved.

Single Intranasal Administration of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine in C57BL/6 Mice Models Early Preclinical Phase of Parkinson`s Disease

Many studies have shown that deficits in olfactory and cognitive functions precede the classical motor symptoms seen in Parkinson`s disease (PD) and that olfactory testing may contribute to the early diagnosis of this disorder. Although the primary cause of PD is still unknown, epidemiological studies have revealed that its incidence is increased in consequence of exposure to certain environmental toxins. In this study, most of the impairments presented by C57BL/6 mice infused with a single intranasal (i.n.) administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (1 mg/nostril) were similar to those observed during the early phase of PD, when a moderate loss of nigral dopamine neurons results in olfactory and memory deficits with no major motor impairments. Such infusion decreased the levels of the enzyme tyrosine hydroxylase in the olfactory bulb, striatum, and substantia nigra by means of apoptotic mechanisms, reducing dopamine concentration in different brain structures such as olfactory bulb, striatum, and prefrontal cortex, but not in the hippocampus. These findings reinforce the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD. These results also provide new insights in experimental models of PD, indicating that the i.n. administration of MPTP represents a valuable mouse model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

Circulating cell-free DNA levels in plasma increase with severity in experimental acute pulmonary thromboembolism

Background: The diagnosis of acute pulmonary thromboembolism (APT) and its severity is challenging. No previous study has examined whether there is a linear relation between plasma DNA concentrations and the severity of APT. We examined this hypothesis in anesthetized dogs. We also examined the changes in plasma DNA concentrations in microspheres lung embolization and whether the therapy of APT with nitrite could modify APT-induced changes in plasma DNA concentrations. In vitro DNA release from blood clots was also studied. Methods: APT was induced with autologous blood clots (saline, 1, 3, or 5 ml/kg) injected into the right atrium. A group of dogs received 300 pm microspheres into the inferior vena cava to produce similar pulmonary hypertension. Another group of dogs received 6.75 mu mol/kg nitrite after APT with blood clots of 5 ml/kg. Hemodynamic evaluations were carried out for 120 min. DNA was extracted from plasma samples using QIAamp DNA Blood Mini Kit and quantified using Quant-iT (TM) PicoGreen (R) dsDNA detection kit at baseline and 120 min after APT. Results: APT produced dose-dependent increases in plasma DNA concentrations. which correlated positively with pulmonary vascular resistance (P=0.002, r=0.897) and with mean pulmonary arterial pressure (P=0.006, r=0.856). Conversely, lung embolization with microspheres produced no significant changes in plasma DNA concentrations. While nitrite attenuated APT-induced pulmonary hypertension, it produced no changes in plasma DNA concentrations. Blood clots released dose-dependent amounts of DNA in vitro. Conclusions: Cell-free DNA concentrations increase in proportion to the severity of APT, probably as a result of increasing amounts of thrombi obstructing the pulmonary vessels. (C) 2009 Elsevier B.V. All rights reserved.

Mice with genetic deletion of the heparin-binding growth factor midkine exhibit early preclinical features of Parkinson`s disease

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson`s disease (PD) begin many years before the appearance of the characteristic motor symptoms and that impairments in olfactory, cognitive and motor functions are associated with time-dependent disruption of dopaminergic neurotransmission in different brain areas. Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in many biological processes in the central nervous system such as cell migration, neurogenesis and tissue repair. The abnormal midkine expression may be associated with neurochemical dysfunction in the dopaminergic system and cognitive impairments in rodents. Here, we employed adult midkine knockout mice (Mdk(-/-)) to further investigate the relevance of midkine in dopaminergic neurotransmission and in olfactory, cognitive and motor functions. Mdk(/-) mice displayed pronounced impairments in their olfactory discrimination ability and short-term social recognition memory with no gross motor alterations. Moreover, the genetic deletion of midkine decreased the expression of the enzyme tyrosine hydroxylase in the substantia nigra reducing partially the levels of dopamine and its metabolites in the olfactory bulb and striatum of mice. These findings indicate that the genetic deletion of midkine causes a partial loss of dopaminergic neurons and depletion of dopamine, resulting in olfactory and memory deficits with no major motor impairments. Therefore, Mdk(-/-) mice may represent a promising animal model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

We examined whether two functional polymorphisms (g.-1562C>T and g.-90(CA)14-24) in the matrix metalloproteinase (MMP)-9 gene or MMP-9 haplotypes affect the circulating levels of pro-MMP-9 and pro-MMP-9/TIMP-1 (tissue inhibitor of metalloproteinase-1) ratios in AIDS patients, and modulate alterations in these biomarkers after highly active antiretroviral therapy (HAART). We studied 82 patients commencing HAART. Higher pro-MMP-9 concentrations and pro-MMP-9/TIMP-1 ratios were found in CT/TT patients compared with CC patients. HAART decreased pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratios in CT/TT patients, it did not modify pro-MMP-9 levels and it increased pro-MMP-9/TIMP-1 ratios in CC patients. The g.-90(CA)14-24 polymorphism, however, produced no significant effects. Moreover, we found no significant differences in HAART-induced changes in plasma pro-MMP-9, TIMP-1 and pro-MMP-9/TIMP-1 ratios when different MMP-9 haplotypes were compared. These findings suggest that the g.-1562C>T polymorphism affects pro-MMP-9 levels in patients with AIDS and modulates the alterations in pro-MMP-9 levels caused by HAART, thus possibly affecting the risk of cardiovascular complications. The Pharmacogenomics Journal (2009) 9, 265-273; doi: 10.1038/tpj.2009.13; published online 21 April 2009

Increased circulating levels of matrix metalloproteinase (MMP)-8, MMP-9, and pro-inflammatory markers in patients with metabolic syndrome

Background: Metabolic syndrome (MetS) predisposes to cardiovascular complications. Increased concentrations of pro-inflammatory mediators and imbalanced concentrations of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) may reflect the pathophysiology of MetS. We compared the circulating levels of MMPs, TIMPs, and inflammatory mediators in MetS patients with those found in healthy controls. Methods: We studied 25 healthy subjects and 25 MetS patients. The plasma levels of pro-MMP-2 and pro-MMP-9 were determined by gelatin zymography. The plasma concentrations of MMP-8, MMP-3, TIMP-1, TIMP-2, monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), intercellular adhesion molecule (sICAM-1), and sP-selectin were measured by ELISA kits. Results: We found higher sP-selectin, sICAM-1, MCP-1, and IL-6 (all P<0.05) concentrations in MetS patients compared with healthy controls. No differences in pro-MMP-2, MMP-3, and TIMP-2 levels were found (all P>0.05). However, we found higher pro-MMP-9, MMP-8. and TIMP-1 levels in MetS patients compared with healthy controls (all P<0.05). Conclusions: Patients with MetS have increased circulating concentrations of pro-MMP-9, MMP-8, and TIMP-1 that are associated with increased concentrations of pro-inflammatory mediators and adhesion molecules. These findings suggest that MMPs may have a role in the increased cardiovascular risk of MetS patients. Pharmacological interventions targeting MMPs, especially MMP-9 and MMP-8 deserve further investigation in MetS patients. (C) 2009 Elsevier B.V. All rights reserved.

Effects of statins on matrix metalloproteinases and their endogenous inhibitors in human endothelial cells

Statins exert anti-inflammatory effects and downregulate matrix metalloproteinases (MMPs) expression, thus contributing to restore cardiovascular homeostasis in cardiovascular diseases. We aimed at comparing the effects of different statins (simvastatin, atorvastatin, and pravastatin) on MMP-2, MMP-9, tissue inhibitors of metalloproteinases (TIMP)-1, TIMP-2, and MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios released by human umbilical vein endothelial cells (HUVEC) stimulated by phorbol myristate acetate (PMA). HUVECs were incubated with statins (0.1-10 mu M) for 12 h before stimulation with PMA 100 nM. Monolayers were used to perform cell viability assays and the supernatants were collected to determine MMPs and TIMPs levels by gelatin zymography and/or enzyme immunoassay. While treatment with PMA increased MMP-9 and TIMP-1 levels (by 556% and 159%, respectively; both P < 0.05), it exerted no effects on MMP-2 and TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, attenuated PMA-induced increases in MMP-9 levels (P < 0.05). Only atorvastatin decreased baseline MMP-2 levels significantly (P < 0.05). We found no effects on TIMP-2 levels. Simvastatin and atorvastatin, but not pravastatin, decreased MMP-9/TIMP-1 ratio significantly (both P < 0.05), whereas atorvastatin and pravastatin, but not simvastatin, decreased MMP-2/TIMP-2 ratio significantly (both P < 0.05). Our data support the notion that statins with different physicochemical features exert variable effects on MMP/TIMP ratios (which reflect net MMP activity). Our results suggest that more lipophilic statins (simvastatin and atorvastatin), but not the hydrophilic statin pravastatin, downregulate net MMP-9 activity. However, atorvastatin and pravastatin may downregulate net MMP-2 activity. The clinical implications of the present findings deserve further investigation.

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane: Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma ), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.