Luciano Artese

Suppression of the functionally coupled cyclooxygenase-2/prostaglandin E synthase as a basis of simvastatin-dependent plaque stabilization in humans.

Background—The clinical benefits of statins are attributed to changes in plaque composition that lead to reduced metalloproteinase (MMP) activity and plaque stabilization. However, the molecular mechanism of this effect is unclear. Recently, we demonstrated enhanced expression of isoforms of inducible cyclooxygenase (COX) and PGE synthase (COX-2/mPGES) in human symptomatic plaque and provided evidence that this is associated with MMP-induced plaque rupture. The aim of this study was to characterize the effect of simvastatin on inflammatory infiltration and the expression of COX-2/mPGES and MMPs in human carotid plaques. Methods and Results—Seventy patients with symptomatic carotid artery stenosis were randomized to the American Heart Association Step 1 diet plus simvastatin (40 mg/d) or the American Heart Association Step 1 diet alone for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunocytochemistry, Western blot, and reverse transcription–polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunocytochemistry was also used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the simvastatin group had fewer (P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells; less (P <0.0001) immunoreactivity for COX-2/mPGES and MMPs; reduced (P <0.0001) gelatinolytic activity; increased (P <0.0001) collagen content; and reduced (P <0.0001) lipid and oxLDL content. Interestingly, COX-2/mPGES inhibition by simvastatin was completely reversed by mevalonate in vitro. Conclusions—This study demonstrates that simvastatin decreases inflammation and inhibits COX-2/mPGES expression in plaque macrophages, and this effect in turn may contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.

Human herpesvirus 8 variants in sarcoid tissues

BACKGROUND The cause of sarcoidosis is unknown, although mycobacteria have been implicated. We examined sarcoid tissues for human herpesvirus 8 (HHV-8) in addition to mycobacterial genomic sequences. METHODS Biopsy samples from 17 patients with sarcoidosis were studied (eight transbronchial, 27 lymph node, two skin, and two oral mucosa). We used tissues (n = 137) from 96 patients without sarcoidosis as negative controls. A nested PCR was applied to amplify a segment of open reading frame (ORF) 26 of the HHV-8 genome, and a heminested PCR was to amplify a segment of ORF 25 of HHV-8 and of the 16 S rRNA gene of mycobacteria. Differences in base sequences of the amplified fragments were resolved with single-strand conformation polymorphism and dideoxy sequencing. FINDINGS HHV-8 ORF 26 DNA was detected in significantly higher proportions of sarcoid than of non-sarcoid tissue samples from lung (8/8 vs 0/54; p < 0.0001), lymph nodes (26/27 vs 6/29; p < 0.0001), skin (2/2 vs 0/17; p = 0.006), and oral tissues (2/2 vs 1/13; p = 0.029). 31 (82%) of the 38 ORF 26 DNA-positive sarcoid specimens were also positive for ORF 25 DNA. For mycobacteria-like 16 S rRNA DNA, the proportion positive was significantly higher in sarcoid than non-sarcoid tissues for lymph node samples (11/27 vs 2/29; p = 0.003) but not for other tissues (lung 3/8 vs 22/54; skin 2/2 vs 15/17; and oral tissues 1/2 vs 0/13). Overall, the prevalence of HHV-8 ORF 26 sequences was higher in sarcoid tissues than in non-sarcoid tissues (p < 0.0001). When patients whose tissues were included in a masked phase of the study were treated as units of analysis, eight of eight sarcoidosis patients were positive for HHV-8 ORF 26 DNA, compared with three of 56 control patients (p < 0.0001); for mycobacteria-like sequences, three of eight sarcoidosis patients were positive, compared with four of 56 controls (p = 0.0464). The HHV-8 ORF 26 sequences, ten of which were unique, could be segregated into four groups according to peptide motifs. In seven of nine patients from whom biopsy samples were taken from various sites, different sequences were recovered. The mycobacterial sequences amplified from sarcoid tissues were also varied, but none was homologous to those of known species. INTERPRETATION Variant HHV-8 DNA sequences are found in a wide range of sarcoid but not non-sarcoid tissues. Mycobacteria-like 16 S rRNA sequences are more frequently present in sarcoid lymph nodes and not in other tissue types, but do not indicate infection by a particular mycobacterial species.

Vascular Endothelial Growth Factor (VEGF) Expression in Healthy and Inflamed Human Dental Pulps

Vascular endothelial growth factor (VEGF) is a glycoprotein that has the capability to increase vascular proliferation and permeability. VEGF has been found to be expressed in several different types of tumors, and it may contribute to the progression of malignant tumors. Immunostaining for VEGF and factor VIII was performed in normal healthy pulps and in irreversible pulpitis. In both cases the vessels were always positive for VEGF. Our immunohistochemical data show that the expression of VEGF was strongly positive in the inflammatory infiltrate in irreversible pulpitis. VEGF expression in the stromal cells in healthy pulps ranged from 20 to 100% (with a mean of 68.82), and in irreversible pulpitis ranged from 0 to 100% (with a mean of 62.35%); this difference was statistically significant (p = 0.05). This down-regulation in the stromal cells in irreversible pulpitis could be due to the presence, in a low compliance system such as the dental pulp, of inflammatory infiltrate. VEGF is probably a factor implicated in the etiology and progression of pulpitis. The microvessel density in healthy pulps was 90.00 +/- 27.5, while, in irreversible pulpitis, it was 56.68 +/- 21.15. This difference was statistically significant (p = 0.001). The decrease in microvessel density in irreversible pulpitis could be related to failing vascular function and blood flow decrease.

Immune cells in periapical granuloma: Morphological and immunohistochemical characterization*

Samples of periapical granulomas obtained from 12 patients were examined using light and electron microscopes and monoclonal antibodies. Monocytes/macrophages, lymphocytes, and plasma cells were nearly always the most abundant cell populations. Ultrastructural analysis showed close contacts between macrophages and cells of the lymphoid lineage, with the lymphoid cells frequently demonstrating blastic features. Immunohistochemical staining with the anti-interleukin 2 receptor antibody showed that the concentration of labeled cells was quite low. The vast majority were lymphocytes, though some mast cells were also labeled. Mast cells were chiefly located in perivascular areas and interleukin 2 receptor-positive mast cells were frequently associated with lymphoid cells. mast cells could be part of a negative feedback mechanism in the immune response. By releasing histamine, they would block the immune response and by absorbing interleukin 2 they would remove it as an immune system stimulant.

Alveolar Ridge Regeneration with Equine Spongy Bone: A Clinical, Histological, and Immunohistochemical Case Series

BACKGROUND In the case of localized ridge atrophy, a ridge augmentation procedure, with the use of bone substitutes and barrier membranes, may then be necessary. PURPOSE The aim of the present study was a clinical, histological, and immunohistochemical evaluation of an equine spongy bone in alveolar ridge augmentation procedures. MATERIALS AND METHODS Five patients showing horizontal mandibular ridge defects participated in this study. A ridge augmentation was performed through an onlay apposition of equine bone covered by a titanium-reinforced membrane. After 6 months of healing, five bone cores from nonaugmented sites (control) and five from augmented sites (test) were retrieved. RESULTS In test sites, no postoperative complications occurred. Horizontal bone width increased from 24 to 37 mm. In control sites, the newly formed bone represented 33%, and in test sites, 35% of the total area. The mean value of the microvessel density was 25.6 +/- 3.425 per mm(2) in controls, while 33.3 +/- 2.5 vessels per mm(2) in the test sites were found (p < .05). Both groups showed a high intensity (++) of vascular endothelial growth factor expression in the newly formed bone, while a low intensity (+) was found in the mature bone. CONCLUSION Equine bone appeared to be biocompatible and to be associated with new vessel ingrowth. Within the limits of the small sample size, the present study indicated that equine bone could be used in mandibular ridge augmentations.

Immunoexpression of angiogenesis, nitric oxide synthase, and proliferation markers in gingival samples of patients with aggressive and chronic periodontitis.

BACKGROUND In periodontal tissues, angiogenesis seems to be important for the maintenance of healthy tissues and in periodontal diseases. Angiogenesis is regulated through a complex interplay of molecular signals mediated by growth factors involving extracellular matrix remodeling, endothelial cell migration and proliferation, capillary differentiation and anastomosis. However, the influence of angiogenesis in the development, progression, and healing of periodontal lesions is currently under investigation. This immunohistochemical study evaluates the expression of vascular endothelial growth factor (VEGF), microvessel density (MVD), nitric oxide synthase (NOS) 1 and 3, and Ki-67 in gingival tissues of patients with aggressive and chronic periodontitis. METHODS Twenty-seven human gingival biopsies were taken from patients with chronic periodontitis (n = 14 patients), generalized aggressive periodontitis (n = 6 patients), and healthy periodontia (n = 7 patients). The specimens were immunohistochemically stained for VEGF, MVD, NOS 1 and 3, and Ki-67. RESULTS The levels of VEGF, MVD, NOS 1 and 3, and Ki-67 were found to be significantly different among groups (P >0.001). Patients with aggressive periodontitis had higher levels of these markers compared to those in patients with chronic periodontitis and healthy patients. CONCLUSIONS The analysis demonstrates a higher expression of all immunologic markers particularly in subjects with aggressive periodontitis. In summary, the data from this pilot investigation suggests that VEGF is an important factor in the pathogenesis of the aggressive and chronic forms of periodontitis.

Microvessel density in sinus augmentation procedures using anorganic bovine bone and autologous bone: 3 months results.

Purpose:This study was an immunohistochemical evaluation of microvessel density (MVD) in sinus augmentation procedures with autologous bone and anorganic bone (Bio-Oss®). Materials and Methods:Twenty-four patients (14 men and 10 women – mean age of 48 years with a range from 34 to 53 years) participated in this study. All the patients presented a maxillary partial unilateral edentulism involving the premolar/molar areas, with a residual alveolar ridge height of about 4 to 5 mm. Twelve patients received sinus augmentation procedures with 100% autologous bone; 100% Bio-Oss was used in the other 12 patients. Endosseous implants were inserted after a mean period of 3 months. As control, the portions of preexisting subantral bone were used. The mean value of the MVD in control bone was 23.4 +/− 1.3. The mean value of the MVD in the sinuses augmented with autologous bone was 29.0 +/− 2.4. The mean value of the MVD in the sinuses augmented with Bio-Oss was 23.8 +/− 2.2. Results:The statistical analysis showed that the differences of the MVD between control bone and sinuses augmented with Bio-Oss were not statistically significant (P = 0.52), while the difference of the MVD between sinuses augmented with autologous bone and those augmented with Bio-Oss was statistically significant (P = 0.0008). Conclusions:Autologous bone may act not only as a passive filling material in bone defects but may also release osteogenic growth factors; and particles of autologous bone seem to contain vital osteoprogenitor cells.

VEGF and MVD expression in sinus augmentation with autologous bone and several graft materials

OBJECTIVE The aim of this study was to assess vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in maxillary sinus augmentation with autogenous bone and different graft materials for evaluating their angiogenic potential. METHODS Biopsies were harvested 10 months after sinus augmentation with a combination of autogenous bone and different graft materials: hydroxyapatite (HA, n = 6 patients), demineralized freeze-dried bone allograft (DFDBA, n = 5 patients), calcium phosphate (CP, n = 5 patients), Ricinus communis polymer (n = 5 patients) and control group--autogenous bone only (n = 13 patients). RESULTS In all the samples, higher intensities of VEGF expression were prevalent in the newly formed bone, while lower intensities of VEGF expression were predominant in the areas of mature bone. The highest intensity of VEGF expression in the newly formed bone was expressed by HA (P < 0.001) and CP in relation to control (P < 0.01) groups. The lowest intensities of VEGF expression in newly formed bone were shown by DFDBA and polymer groups (P < 0.05). When comparing the different grafting materials, higher MVD were found in the newly formed bone around control, HA and CP (P < 0.001). CONCLUSION Various graft materials could be successfully used for sinus floor augmentation; however, the interactions between bone formation and angiogenesis remain to be fully characterized.

Hemostasis control in endodontic surgery: a comparative study of calcium sulfate versus gauzes and versus ferric sulfate.

INTRODUCTION Calcium sulfate (CaS) is a simple, biocompatible material with a long history of safe use in different fields of medicine. CaS is a rapidly resorbing material that leaves behind a calcium phosphate lattice, which promotes bone regeneration and hemostasis. The aim of this study was a clinical evaluation of the hemostatic effect of CaS hemi-hydrate (CaSO4), commonly known as plaster of Paris, in endodontic surgery. METHODS Twenty-four patients with 31 periradicular lesions were enrolled in this study. The apical roots were exposed, and the bleeding would have made it difficult to correctly fill the root-end cavities. To avoid such an inconvenience, the teeth were divided into 3 groups. Hemostasis was attempted by using CaS in 11 teeth (group I), gauze tamponade in another 10 teeth (group II), or 20% ferric sulfate in the last 10 teeth (group III). RESULTS Control of the bleeding was achieved in all teeth of group I, whereas in group II adequate hemostasis was achieved in 3 of 10 cases and in group III in 6 of 10 cases. CONCLUSIONS The use of CaS completely eliminated the bleeding, with a very good level of hemostasis.

Histological and immunohistochemical evaluation of the peri-implant soft tissues around machined and acid-etched titanium healing abutments: a prospective randomised study

A close spatial correlation has been described between the roughness of intraoral materials and the rate of bacterial colonisation. The aim of the present study in man was to conduct a comparative immunohistochemical evaluation of the inflammatory infiltrate, microvessel density, the nitric oxide synthases 1 and 3 and the vascular endothelial growth factor expression, the proliferative activity, and the B and T lymphocyte and histiocyte positivity in the peri-implant soft tissues around machined and acid-etched titanium healing caps. Ten patients participated in this study. The patients were enrolled consecutively. All patients received dental implants left to heal in a non-submerged mode. Healing caps were inserted in all implants. Half of the implants were supplied randomly with machined caps of titanium (control), while the other half were provided randomly with acid-etched titanium caps (test). After a 6-month healing period, a gingival biopsy was performed with a circular scalpel around the healing caps of both groups. The inflammatory infiltrate was mostly present in test specimens. Their extension was much larger than that of the control samples. A higher number of T and B lymphocytes were observed in test specimens. Higher values of microvessel density and a higher expression of vascular endothelial growth factor intensity were observed in the test samples. Furthermore, the Ki-67, NOS1 and NOS3 expression was significantly higher in the test specimens. All these results showed that the tissues around test healing caps underwent a higher rate of restorative processes, most probably correlated to the higher inflammation processes observed in these tissues.