Immunohistochemical study of osteopontin in oral squamous cell carcinoma allied to fractal dimension.

The aim of the study was to consider a possible correlation between the intensity of expression of osteopontin and grading established by the pathologist. Furthermore, a correlation was investigated between the increase of fractal dimension and osteopontin in order to use this marker as an early and reliable diagnostic tool for the degree of cell transformation in oral squamous carcinoma. Ten histologically healthy oral samples and sixty-four primary oral squamous cell carcinomas specimens were analysed by a single pathologist. Immunohistochemical analysis and Fulgen stain were performed in order to evaluate intensity of expression of osteopontin and fractal dimension. Data obtained were presented as mean and standard deviation and processed for the statistical analysis. Ostepontin expression revealed a statistical significance between groups (P less than 0.001). Fractal dimension in oral squamous cell carcinoma groups vs controls revealed statistically significant differences (P less than 0.001). The fractal dimension value and the osteopontin expression were compared, using two-dimensional scatter. The correlation was relevant in the G3 group. The results demonstrated a correlation between the growths of osteopontin expression and nuclear abnormality measured by fractal dimension. These results support the hypothesis that the level of osteopontin expression might be used as a marker for the evaluation of oral squamous cell carcinoma differentiation. Osteopontin and fractal dimension could support the histological grading to increase the predictability of the diagnosis, choices of treatment procedure and long-term prognosis.

Role of matrix metalloproteinases in radicular cysts and periapical granulomas.

AIM: The aim of the present study was to evaluate the expression and distribution of different classes of matrix metalloproteinases (MMPs) in radicular cysts and periapical granulomas. METHODS: Twenty consecutive specimens of radicular cysts and 20 of periapical granulomas were selected. Expression of MMP-2, -9, -8, -13, -3 was immunohistochemically evaluated. The intensity of expression of the MMPs was evaluated using a semi-quantitative analysis: low = +; intermediate = ++; high = +++. RESULTS: Positive expression of MMPs was present with different distribution. MMP-9 expressed differently in the lesions. Indeed, in periapical granulomas low expression was found in endothelial cells and fibroblasts, whilst high intensities were only detected in inflammatory cells. On the contrary, in radicular cysts the high intensities were mainly present in keratinocytes and fibroblasts. MMP-8 was mainly expressed in inflammatory cells of periapical granulomas. MMP-2 and -3 presented a low intensity of expression in both groups. MMP-13 showed a variable pattern of distribution in the different cell types of the two different lesions. CONCLUSION: The present investigation supports the role of MMPs in the inflammatory process leading to the development of radicular cysts and periapical granulomas. The results of the present study suggested that the increased enlargement of radicular cysts, compared to periapical granulomas, might be related to a higher expression of MMP-9. On the other hands, the higher intensity of expression of MMP-8 in periapical granulomas could be related to an active inflammatory process. MMP-8 could play an important role in the inflammation processes during the development of periapical lesions.

Immunoexpression of angiogenesis and proliferation markers in patients treated with cyclosporin A.

AIM: In the present immunohistochemical study, the expression of vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67 in the gingival tissues of renal transplant patients treated with cyclosporin A was assessed. Gingival overgrowth (GO) frequently occurs in transplant patients receiving immunosuppressive drugs such as cyclosporine and this gingival inflammation might play an important role in the pathogenesis of drug-induced GO. METHODS: Twenty-eight human gingival biopsies were taken from healthy patients with chronic periodontitis (N.=14 control group), and from renal transplant recipients treated with cyclosporin A (N.=14 test group). The retrieved specimens were immunohistochemically processed and stained for vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67. RESULTS: The levels of vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67 were found to be significantly different among groups (P>0.001), with patients treated with cyclosporin A showing higher levels of all the analyzed markers compared to control group. CONCLUSION: In summary, the data from this pilot study suggests that the investigated factors have a role in the inflammation processes associated to immunosuppressive therapy. However, further studies with a larger sample population need to be conducted for an exhaustive knowledge of the mechanisms leading to GO.

Thyroid cysts treatment with the sclerosing agent Atossiclerol. Long-term (5 to 15 years) follow-up.

AIM: The aim of this study was to evaluate the long-term efficacy of the sclerosing agent Atossisclerol in the treatment of benign, ecolucent, thyroid cysts. METHODS: Two groups of cysts, one between 1 and 3 cm and a group smaller than 0.99 mm in maximum diameter (range 0.6-0.99) were considered in the inclusion plan. The localization of the cysts was at lobar level. A limited quantity of Atossisclerol (0.5 to 2%) was injected. RESULTS: No side effects were noted. After 5 years in the group treated with the sclerosing agent 93% of the cysts were completely disappeared (vs 60% in controls). However the initial target cyst at 5 and years was completely cured in most patients (>80%) treated with the sclerosing agent; at 10 years more than 90% of the original, sclerosed target cysts were not visible. Another control of most of these patients (53/68) at 15 years indicated that sclerosis of the cyst is safe and allow a better occlusion/obliteration (in almost all patients) of the cyst in comparison with aspiration only (slow recurrence in some 25% of the cysts). CONCLUSION: The sclerosing agent can be re-injected in the same patients without significant reaction or clinical problems or immunological reactions.

Management of compound odontoma in a 10-year-old girl preserving the associated impacted permanent tooth.

AIM: Compound odontoma has been reported to be the most common of all odontogenic neoplasms and tumor- like lesions. Only rarely the treatment of this lesion in association with an impacted tooth has been reported. CASE REPORT: A compound odontoma in a 10-year-old girl, associated with an impacted permanent incisor is described, focusing on the diagnosis and the importance of early treatment of this lesion. The patient underwent surgical excision of the lesion and it was decided to wait for the spontaneous eruption of the impacted tooth. After 6 months no eruption was observed and thus the orthodontic treatment was deemed necessary. At the one-year follow-up, the tooth was brought into the maxillary arch.

Morphometric and ultrastructural analysis of human granulosa cells after gonadotrophin-releasing hormone agonist or antagonist.

Morphological features of granulosa cells can reflect their functional status. The present study was aimed at comparing possible differences in the fine structure of human granulosa cells exposed to gonadotrophin-releasing hormone (GnRH) agonist or antagonist treatment during ovarian stimulation. Cells were obtained from follicular aspirates of 21 women treated with recombinant follicle-stimulating hormone (rFSH) plus either a GnRH agonist or a GnRH antagonist. Conventional light microscopy procedures and computerized image analysis systems were used to identify different cell type morphological patterns and to quantify different cells distribution. Two morphologically distinct granulosa cell populations, defined as large/pale and small/dark cells, were identified and a different distribution in the two groups of women under investigation was found: a significantly higher percentage in large/pale cells was detected in the agonist-treated women (P<0.05), whereas the percentage of small/dark cells was significantly higher in the antagonist-treated group (P<0.05). Ultrastructural observations showed the presence in both cell populations of typical hallmarks of steroidogenic cells, highlighting signs of functional activity in the large/pale cell population. Further investigations are needed to define the possible clinical significance of these morphological findings.

Neuroglobin in aging carotid bodies.

Neuroglobin (Ngb) is a member of the vertebrate globin family expressed particularly in the brain and in the retina. Ngb is concentrated in the mitochondria-containing areas of neurons, and its distribution is correlated with oxygen consumption rates. Previously we have shown that Ngb is expressed in carotid body (CB) tissues. Considering that hypoxia and aging may be linked through a series of adaptive and protective mechanisms (e.g. reduction in mitochondrial numbers), we investigate the role of Ngb during aging and hypoxia. Two groups of six rats (age-matched 3 and 24 months old) were kept in room air as a control groups, the others two groups were kept in a Plexiglas chamber for 12 days in chronic hypoxia (10-12% inspired oxygen). The presence of Ngb in the CB tissue was detected by immunohistochemistry using a polyclonal antibody. Ngb immunoreactivity was significantly higher in CB tissues from young rats exposed to chronic intermittent hypoxia, whereas CB tissues from old rats did not show any significant increase in Ngb levels after hypoxia. Similar to hemoglobin, Ngb may act as a respiratory protein by reversibly binding gaseous ligands NO and O(2) and could act as a NO scavenger and participate in detoxification of Reactive Oxygen Species (ROS) generated under hypoxic conditions.

Aging and expression of heme oxygenase-1 and endothelin-1 in the rat carotid body after chronic hypoxia.

Hypoxia transiently increases transcription of the gene encoding heme oxygenase-1 (HO-1) and potently activates production of endothelin-1 (ET-1), the latter of which plays a central role in cellular adaptation to hypoxia. The ventilatory response to hypoxia attenuates with aging, and decreased responsiveness to hypoxia is seen in the aged vs. young rats, suggesting that the functionality of the oxygen-sensitive mechanism is age-dependent. In the present study, we examined the effects of aging on the expression of HO-1 and ET-1 in the carotid body, which is a small cluster of chemoreceptors and supporting cells that measure changes in the composition of arterial blood flowing through it. Our results revealed that HO-1 and ET-1 were expressed in carotid bodies of both young and old rats, although less so in the old ones. Exposure to chronic intermittent hypoxia significantly increased both HO-1 and ET-1 immunoreactivity in both young and old carotid body tissues, with the persisting age-dependent inequality to the disadvantage of old age. Considering that ET-1 is capable of enhancing intermittent hypoxia-induced chemosensory responses by the carotid body, our results suggest that decreased induction of ET-1 and HO-1 during aging could form the basis for age-related reductions in chemosensory discharge.

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.

Peripheral giant cell granuloma associated with a dental implant. A case report.

Peripheral giant cell granuloma (PGCG) is a tumor-like pathologic condition arising on the buccal or lingual attached gingival or alveolar mucosa and the crest of the edentulous alveolar ridge, of uncertain etiology; it is probably a reactive lesion caused by chronic local irritants or trauma rather than a true neoplasm. PGCG is thought to originate from elements of the periodontal ligament or from the periosteum. Clinically, it appears as a sessile or broadly pedunculated, bluish to purple-red, fleshy or firm swelling with a frequently ulcerated surface. The occurrence of such a lesion may be significant for the prognosis of dental implants, and they can lead to integration failure. Treatment of choice is conservative surgical excision with total removal of the base of the lesion and with curettage of the underlying bone. However, some of these lesions recur. Clinically, it may be difficult to distinguidsh PGCG from pyogenic granuloma. Moreover, also peripheral odontogenic tumors have to be considered in the differential diagnosis of PGCG. The clinical appearance of all these lesions are similar and so the definitive diagnosis is only histological and a biopsy with micreoscopical examination is mandatory.

Vascular endothelial growth factor expression (VEGF) in salivary glands of diabetic rats.

This study is aimed at evaluating vascular endothelial growth factor (VEGF) expression in the salivary glands of diabetic rats. Our study was carried out on 1 group of male Wistar rats and 1 group of male Bio Breeding Wistar (BB/W) pre-diabetic rats. The first group (control) was composed of 11 rats (4 months old), and the other (test) of 11 rats (4 months old) which showed diabetes at day 85 of life. This kind of diabetes can be compared to human diabetes mellitus type 1. The submandibular glands were removed and processed for immunohistochemical analysis of VEGF. Diabetes increased salivary gland VEGF expression in the rats. The tissues analyzed (vascular endothelium, ductal endothelium, mucinous glands) always expressed VEGF, thus demonstrating that not only vascular endothelial cells, but also the other elements evaluated, have a role in the neoangiogenesis process. In both control and diabetic rats, the VEGF expression was constantly negative only in serous acini; thus demonstrating that serous acini are not involved in the neoangiogenetic processes. The vascular growth is a fundamental part of normal salivary gland development, therefore we speculate that strategies aimed at preservation or promotion of salivary gland VEGF expression may mitigate or attenuate diabetic-induced gland microvascular injury.

MIB-1, Bcl-2 and p53 in odontogenic myxomas of the jaws.

Odontogenic myxoma is a rare benign neoplasm occurring in the jaws. Microscopically, it is composed of spindle or stellate-shaped cells arranged in a mucinous matrix. In some cases (20%), odontogenic epithelial islands may be found. The Authors evaluated p53, MIB-1, and Bcl-2 expressed by the epithelial and stromal elements in 12 cases of odontogenic myxoma of the jaws. The cells of the odontogenic epithelium were positive for Bcl-2, p53 and MIB-1. The stromal cell showed a high positivity for MIB-1. Proliferation of both the epithelial and stromal components could be related to the growth of this odontogenic tumour.

Microvessel density and vascular endothelial growth factor expression in sinus augmentation using Bio-Oss.

AIM: The aim of this study was to evaluate microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression in sinus augmentation with Bio-Oss. METHODS: Twenty patients participated in this study. The sinuses were filled with 100% Bio-Oss. Implants were inserted after 3 months in group A, and 6 months in group B. A trephine was used to harvest bone cores. As control, the pre-existing subantral bone was used. RESULTS: The mean MVD in control bone was 23.6 +/- 1.8. In the sites augmented with Bio-Oss, at 3 months, the MVD was 23.3 +/- 2.1, while in the sites retrieved at 6 months the MVD was 29.5 +/- 2.4. The difference in MVD between the control bone and group A was not statistically significant. The difference between the control bone and group B was statistically significant (P < 0.05). The statistical analysis showed that the difference in MVD between group A and group B was statistically significant (P < 0.05). CONCLUSIONS: Bio-Oss seemed to induce an increase in MVD that reached a higher value after 6 months. The percentage of vessels positive to VEGF was higher in group A than in group B. Our data also showed a higher percentage of vessel and stromal cells positive to VEGF and higher MVD values in areas where there was newly formed bone compared with areas where maturation processes were occuring, and this fact could point to a close spatial relationship between angiogenesis and osteogenesis.

Vascular endothelial growth factor (VEGF) in human tooth germ center.

Many oncogenis and tumour suppressor genes found inside normal and pathological cells are fundamental for the processes of development, proliferation and tissue differentiation. The purpose of our study is to show the presence and a possible relationship of the VEGF protein during different phases of the development of human dental germ centers. After cephalometric investigation in 8 orthodontic patients with a mean age of 13 years, (4 females and 4 males), hyperdivergence of the third molars were extracted. The 40 surgical samples were tested with monoclonal human anti-VEGFs antibodies carrying out a semi-quantitative analysis to look for a positive reaction. Reaction for anti-VEGF antibodies was detected in normal embryological tissues and in microvessels near odontogenic cells. During different phases of embryologic development of the dental bud our search showed intracytoplasmatic positive immunoreactions both in the ameloblastic and odontoblastic cells. Additionally, a positive reaction was observed for the VEGF protein in the cells of the stellate reticulum and in those endothelial tissue surrounding the microvessels in all the samples examined.

Factors associated with apoptosis in symptomatic and asymptomatic carotid atherosclerotic plaques.

The aim of this study was to investigate the differences that are present between apoptosis in symptomatic (with symptoms of cerebral ischemic attack) and asymptomatic carotid atherosclerotic plaques. The apoptotic process in macrophages and smooth muscle cells was evaluated. Cellular markers and products of immune cells in symptomatic and asymptomatic atherosclerotic plaque and endoarterectomy specimen were analyzed by immunohistochemistry. No statistically significant differences were present regarding the mean SMC actin-positive area. Using double staining of alpha-smooth muscle actin and TUNEL techniques, the number of smooth muscle cells in apoptosis was statistically higher in symptomatic plaque as compared with asymptomatic plaque. Statistically significant differences (p=0.009) were also found in the CD45-positive cells in the inflammatory infiltrate. The CD68-positive macrophages showed statistically significant differences (p=0.0001). Similarly, the double staining with CD68 and TUNEL revealed that apoptotic macrophages were mainly present in asymptomatic plaques rather than symptomatic plaques. Statistically significant differences (p<0.001) were found in the Bcl-2 expression, with higher values in asymptomatic plaques. Our data showed that the increase of the inflammatory cells contributes to plaque instability and that death due to apoptosis of smooth muscle cells in symptomatic plaques could contribute to their destabilization and explains their tendency to fracture.

Beneficial effects of Batimastat (BB-94), a matrix metalloproteinase inhibitor, in rat experimental colitis.

BACKGROUND AND AIMS: Matrix metalloproteinases (MMPs) represent a group of enzymes that regulate cell-matrix composition playing a major role in the inflammatory response. In the present study we evaluated the ability of the MMP inhibitor Batimastat (BB-94) to modify the course of experimental colitis induced in the rat by trinitrobenzensulfonic acid (TNB). METHODS: Colitis was induced in 40 rats by intracolonic administration of TNB. Animals were divided into four groups of ten rats each: group 1 received only intracolonic TNB, group 2 received TNB+5 mg/kg intraperitoneal BB-94, group 3 TNB+10 mg/kg BB-94 and group 4 TNB+20 mg/kg BB-94. The MMP inhibitor was administered 30 min before induction of colitis and twice daily until death. Ten rats receiving only intracolonic 0.9% saline served as controls. Animals were killed after seven days; segments of colon were removed and used for histological score of inflammation and myeloperoxidase (MPO) activity. RESULTS: Rats receiving only intracolonic 0.9% saline showed no evidence of colitis. The inflammation score was 0.9, MPO activity 0.235 U/mg. Group 1 (TNB-treated rats) exhibited a high inflammation score (12.4) and MPO activity (0.715 U/mg). Conversely, BB-94-treated rats showed, compared to the TNB group, a significantly lower inflammation score and MPO activity in a dose-dependent fashion. Group 2: inflammatory score 10.1, MPO activity 0.474 (p < 0.05 vs. TNB); group 3: inflammatory score 8.3, MPO activity 0.287 (p < 0.01 vs. TNB); group 4: inflammatory score 5.0, MPO activity 0.256 (p < 0.01 vs. TNB). CONCLUSIONS: Treatment with BB-94 has dose-dependent beneficial effects on the inflammatory alterations in rat experimental colitis. Thus, the inhibition of MMPs may represent a novel therapeutic approach for treatment of intestinal inflammation.

Vascular endothelial growth factor and microvessel density around healthy and failing dental implants.

Inflammatory infiltrate may be important in the evolution of inflammatory processes involving peri-implant tissues. Angiogenesis is an important feature of inflammation and healing, but its role in the development and progression or in the healing of periodontal lesions has not been elucidated. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell proliferation. The aim of the present study was to conduct a comparative immunohistochemical evaluation of VEGF and microvessel density (MVD) in normal keratinized gingiva and in peri-implant soft tissues surrounding failing implants. Fifteen patients participated in this study. Ten biopsies were taken from healthy keratinized gingiva, and 10 were taken from peri-implant soft tissues surrounding failing non-submerged implants. In healthy sites, the endothelial lining cells of the vessels always tested positive for VEGF; also, VEGF intensity was high in most cases. Stromal cells were positive for VEGF in 70% to 90% of samples. The MVD was 60.250 +/- 5.123. In peri-implantitis samples, the cells of the inflammatory infiltrate were positive for VEGF in 80% to 100% of cases, and the VEGF intensity was low in all cases. The stromal cells were positive for VEGF in 90% to 100% of cases, and in most cases the intensity was low. The MVD was 101.800 +/- 11.256. The difference in MVD between healthy sites and peri-implantitis was statistically significant (P = .0158). Expression of VEGF was lower in peri-implantitis samples, and this difference was statistically significant (P = .0373). Because of its extensive presence, VEGF is probably a factor in both the maintenance of periodontal physiology and in the progression of peri-implant inflammatory disease.