Involvement of TLR9 in priming the immune response in oral papillomatosis induced by low-risk HPV.

Oral papillomatosis represents a benign lesion of the oral mucosa often induced by human papillomavirus (HPV) or having a non-infection local or general etiology. HPVs are very well adapted and efficient viruses able to produce changes in the immune system, endowed with the ability to replicate in the keratinocytes and to remain silent. The natural evolution of HPV infection is different, depending on the efficiency of the innate immune system. The purpose of this study was to explore Toll-like receptor 9 (TLR9) immunohistochemical expression in low-risk (LR)-HPV oral infection and its ability to facilitate an efficient immune response by activating the macrophages, which serve as main antigen-presenting cells. Samples of two groups of oral mucosae - LR-HPV-positive and HPV-negative - were processed for immunohistochemistry technique and incubated with antibody against TLR9 and cluster of differentiation 68 (CD68). Image analysis and morphometry were conducted to assess the intensity of TLR9 immune signal in the epithelium and the number of macrophages labeled by CD68. We found a statistically significant difference between macrophage count for the subjects in HPV-positive and HPV-negative groups; thought no significant differences of TLR9 immune signal was noted, which demonstrates a diminished immune response in HPV-positive group, probably influencing the time of lesion's clearance.

Variations of Serum Oxidative Stress Biomarkers under First-Line Antituberculosis Treatment: A Pilot Study.

Tuberculosis (TB) is one of the highest infectious burdens worldwide, and pathogenesis is yet incompletely elucidated. Bacilli dissemination is due to poor antioxidant defense mechanisms and intensified oxidative stress. There are few recent studies that analyzed and compared free radicals or antioxidant status before and after anti-TB treatment. Hence, the present study underlines the need to identify oxidative stress as it could be a useful tool in TB monitorisation. Thirty newly diagnosed patients with pulmonary TB were included after signing an informed consent. Blood was collected before receiving first-line anti-tubercular therapy (T0) and after 60 days (T2). Spectrophotometric methods were used to quantify oxidative parameters (TBARS-thiobarbituric acid reactive species); enzymatic antioxidants such as SOD (superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase), and TAC (total antioxidant capacity); and non-enzymatic antioxidants such as GSH (reduced glutathione). A moderate positive correlation was found between GSH and TAC (r = 0.63, p-value = 0.046) and GSH and SOD (r = 0.64, p-value = 0.041) at T2. Increased values of GSH, CAT, and SOD were noted at T2 in comparison with T0, while GPx, TAC, and TBARS decreased at T2. A better monitorisation in TB could be based on oxidative stress and antioxidant status. Nevertheless, restoring redox host balance could reduce TB progression.

Hepatoprotective effect of Syringae vulgaris flos ethanolic extracts in streptozotocin-induced diabetes in rats.

Taking account of increasing world population life expectancy, health services will face with a large number of elderly people with chronic age-related diseases. It has been established that chronic diseases are usually accompanied by oxidative stress induced by the overproduction of reactive oxygen species damaging cellular constituents, under conditions of weakening antioxidant defense systems. The balance between free radicals and antioxidant endogenous systems has a defining role in preventing the damage of macromolecules. In addition to the enzymatic (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase) and non-enzymatic (vitamins A, C, E) endogenous systems, a good source of natural antioxidants are medicinal herbs products or phytochemical compounds. The aim of this study is to evaluate the hepatoprotective effect of Syringae vulgaris flos ethanolic extracts in a rat model of streptozotocin-induced diabetes.

Immunoexpression of vascular endothelial growth factor in gingival mucosa with papilloma and condyloma acuminata.

UNLABELLED: The histological changes of the oral mucosa in contact with a metal alloy dentures is one of the current issues widely debated in the literature. AIM: To highlight the expression of vascular endothelial growth factor (VEGF) in human paraprosthetic gingival mucosa exposed to nickel and copper compounds using the immunohistochemical technique. The selected participants were wearers of fixed dentures made of nickel-based alloys and copper-based alloys. The gingival mucosa fragments were prelevated through excision after removing fixed denture and extraction one of its affected teeth. The gingival mucosa fragments were processed through the histological technique of paraffin inclusion. The paraffin-embedded tissue sections were usually stained with Hematoxylin-Eosin and processed by immunohistochemical technique with VEGF antibody. The gingival mucosa fragments from nickel-based alloys dentures wearers were diagnosed with papilloma and, also, gingival mucosa samples prelevated from copper-based alloys dentures wearers were diagnosed with condyloma acuminata. Immunohistochemical reaction for VEGF was different in the gingival mucosa fragments with papilloma compared with condyloma acuminata samples. In papillomatosis gingival mucosa fragments, VEGF was implicated in principal in vasodilatation and inflammation process, and secondary in angiogenesis. In gingival mucosa fragments with condyloma acuminata, the principal role of VEGF was in angiogenesis and secondary in inflammation.

Immunohistochemical aspects of apoptosis in gingival mucosa with papilloma and condyloma acuminata.

The oral mucosa is a component of the oral ecosystem, which can be aggressed by corrosion products released from the dental alloys used in prosthetic dentistry therapy. The purpose of this study was to compare the in vivo effect of nickel and copper compounds on the oral mucosa cells, including their ability to induce cell death, by analyzing the cytochrome c (cyt. c) immunohistochemical expression. Gingival mucosa fragments obtained from the subjects with dentures manufactured by nickel or copper casting alloys were processed through the histological technique of paraffin inclusion. The sections obtained were stained by usually histological methods in order to highlight the histopathological lesions and also analyzed using the immunohistochemical technique in order to study the cyt. c expression. The papillomatosis lesions were observed in the gingival mucosa fragments obtained from the subjects with nickel-based alloy dentures and the condyloma acuminata lesions were observed in those obtained from the subjects with copper-based alloy dentures. The cyt. c immunohistochemical expression was different in the epithelial layer of two types of mucosal fragments but it was the same in their lamina propria connective tissue. We can conclude that the two types of metal alloys have different effects on the adjacent gingival mucosa.

Heterogeneity of collagen secreting cells in gingival fibrosis--an immunohistochemical assessment and a review of the literature.

AIM: In this work, we compared the histological features of the gingival lesions clinically diagnosed as fibrotic overgrowths due to various etiologic factors as well as an immunohistochemical assessment of fibroblasts phenotypic heterogeneity using the specific labeling for vimentin, α-smooth muscle actin (α-SMA) and fibroblast specific protein-1 (FSP1). MATERIALS AND METHODS: Tissue samples were obtained from 12 patients clinically diagnosed with fibrotic gingival overgrowth, divided in four groups. Fragments of gingiva were processed for paraffin embedding. Serial sections were used for routine staining Hematoxylin-Eosin, trichromic Masson and Goldner-Szekely, and for immunohistochemical reactions to label vimentin, α-SMA and FSP1 using for signal amplification several techniques (EnVision, LSAB, ABC). RESULTS: Storage of collagen fibers, increase of fibroblast number and frequent presence of inflammatory infiltrate are histological issues of all fibrotic gingival overgrowth. The incidence of granulation tissue varies but the frequency of its presence point the attention to the involvement in collagen metabolism imbalance. Immunostaining for vimentin showed a difference between its expression in samples from different groups. Except the cases of fibrosis induced by orthodontic devices, cells positive for α-SMA were rare. FSP1-positive fibroblasts were the most frequent in all cases from all the groups selected for this study. CONCLUSIONS: The phenotype of fibroblasts is different in gingival fibrosis in relation to the risk factor, at present the most common being vimentin-positive and FSP1-positive fibroblasts. Myofibroblasts are rare in gingival fibrosis, the most numerous being in local lesions caused by wearing orthodontic devices and in syndromic fibromatosis. Further studies are required to elucidate the manner in which the active fibroblasts are recruited in relation to the etiologic factor of gingival overgrowth.

Effects of therapy with two combinations of antibiotics on the imbalance of MMP-2÷TIMP-2 in chronic periodontitis.

UNLABELLED: Periodontitis represents a chronic bacterial infection that induces immuno-inflammatory conditions affecting gingiva and tooth-supporting tissues. The role of some biological mediators in periodontal disease was widely investigated, especially that of MMP-8 and MMP-9. Recently, MMP-2 was also considered to be an appropriate therapeutic target for prevention of periodontal disease progression. However, effects of the combination of metronidazole with amoxicillin or spiramycin on the release and activation of MMP-2 and the balance MMP-2÷TIMP-2 were rarely studied. This study was designed to assess the influence of two combinations of antibiotics used for treatment of periodontitis on the balance MMP-2÷TIMP-2. Gingival samples obtained from patients with no pharmacological treated chronic periodontitis and those receiving either the association between amoxicillin-metronidazole and spyramicin-metronidazole were processed for paraffin embedding and then used to perform immunohistochemical reactions in order to detect MMP-2 and TIMP-2. All subjects were evaluated clinically and radiographic at the first visit and after treatment completed, the Loe & Silnees gingival index at six sites per tooth for the whole mouth being recorded. Statistical analysis was performed using non-parametrical techniques. Gingiva samples from untreated chronic periodontitis patients revealed a diffuse positive reaction for MMP-2 in the epithelium and also in fibroblasts and macrophages from the lamina propria. For gingiva samples from patients treated with antibiotics, MMP-2 positive reaction was restricted to deep epithelial layers and few cells of the connective tissue. No significant difference was observed for TIMP-2 expression. The clinical indexes were in accordance with immunohistochemical results. After treatment, gingival index values were significantly lower then before (p<0.001) in both groups treated with antibiotics. CONCLUSIONS: The two combinations of antibiotics tested in our study seem to have a dual ability to reduce inflammation as well as to inhibit MMP-2 activity.

Inducible nitric oxide synthase expression (iNOS) in chronic viral hepatitis and its correlation with liver fibrosis.

BACKGROUND: Nitric oxide (NO) production by the action of the inducible nitric oxide synthase (iNOS or NOS2) is increased in tissues that are stimulated by cytokine and endotoxins. The role of NO in the pathogenesis of chronic viral hepatitis is not fully understood but it seems that its overproduction is responsible for the pathological changes under inflammatory conditions. AIM: In this paper, we analyzed the correlation between immunohistochemical expression of iNOS and liver fibrosis in chronic viral hepatitis. MATERIALS AND METHODS: Liver biopsies from patients diagnosed with chronic viral hepatitis B and C were embedded in paraffin and further used for histological staining and immunohistochemical reactions to detect the expression of iNOS and TGF-ß1. The degree of liver fibrosis was established using special staining (trichromic Masson and Gömöri's silver impregnation). RESULTS: In samples with low degree of fibrosis, we observed a discrete positivity for iNOS in periportal hepatocytes and the immunohistochemical reaction for TGF-ß1 were limited to the endothelial cells of liver sinusoids and pro-inflammatory cells from the portal tracts. Positive reaction for TGF-ß1 increased with the degree of liver fibrosis, while the expression of iNOS was enhanced in hepatocytes, as well as in bile ducts and endothelial cells. CONCLUSIONS: Infection with hepatitis B and C viruses induces iNOS expression in hepatocytes, suggesting that NO overproduction might have an important role in progression of chronic viral hepatitis to cirrhosis.

Role of transforming growth factor ß-connective tissue growth factor pathway in dihydropyridine calcium channel blockers-induced gingival overgrowth.

BACKGROUND: Gingival overgrowth was reported as a side effect after chronic administration of several drugs, which, despite their different pharmacological effect, seem to have the gingival mucosa as a secondary target. The thickness of the gingival epithelium and fibrosis in the lamina propria are unspecific changes that together determine the enlargement of the gingival mucosa, but the molecular mechanisms responsible for the imbalance of collagen synthesis/breakdown are still uncertain. The aim of our study was to assess the role of TGF-ß1-CTGF pathway in the activation of cells with a fibrilogenetic phenotype responsible for the gingival fibrosis developed after chronic administration of dihydropyridine calcium channel blockers. MATERIALS AND METHODS: Fragments of gingival tissue collected from patients clinically diagnosed with gingival overgrowth after chronic administration of nifedipine and amlodipine were processed for paraffin embedding. Serial sections were used for routine staining Masson and Gömöri's silver impregnation in order to reveal collagen accumulation and for immunohistochemical reactions to label TGF-ß1, CTGF, Ki67 and α-SMA. RESULTS: Routine histological staining for collagen revealed the presence of gingival fibrosis and a change between type I collagen/type III collagen ratio. Regardless of the drug involved, many slides showed extended TGF-ß1 positive areas, mainly in the profound - spinous and basal - layers, but also in some cells from the subjacent connective tissue. CTGF exposed intense positive reaction in the basal and parabasal layers, but also in resident cells from the connective tissue. Ki67 immunolabeling did not reveal an increased fibroblast proliferation in the lamina propria. We noticed the presence of a small number of myofibroblasts in the lamina propria. CONCLUSIONS: These findings suggest that TGF-ß1-CTGF axis is activated in dihydropyridine calcium channel blockers-induced gingival overgrowth and exerts a different control on the activation of fibroblasts with a synthetic phenotype. These results also have implications for better understanding mechanisms of fibrosis and the future use of this pathogenic pathway as a therapeutic target in order to limit gingival fibrosis.

Indirect pulp capping in young patients: immunohistological study of pulp-dentin complex.

Indirect capping is a complex therapy exclusively needed in deep cavities that provides, using biomaterials, a disinfection of the dentinary sore and seals the dentinary tubules, protects the pulp of physical mechanisms and chemical agents and stimulates the mechanisms that produce new dentin. Following this idea, we studied the histological changes in the dental pulp tissue and also the specific immunohistochemical response in various structures when an indirect capping technique was used. We used special histological techniques followed by classical staining or by immunohistochemical reaction in order to assess the odontoblastic, and the vascular reaction. The immunohistochemical study allows us to evaluate the changes in the pulp-dentin complex, as the result of the changes in the dentinal tubules permeability and the biological reactions at this level.

Role of innate immune receptors TLR2 and TLR4 as mediators of the inflammatory reaction in human visceral adipose tissue.

White adipose tissue from different locations is characterized by significant differences in the structure of adipocyte "secretoma". Fat accumulation in the central-visceral depots is usually associated with a chronic inflammatory state, which is complicated by the metabolic syndrome. Recently, the adipose tissue was emerged to have an essential role in the innate immunity, adipocytes being considered effector cells due to the presence of the Toll-like receptors (TLRs). In this study, we compared the expression of TNF-α, TLR2 and TLR4 in peripheral-subcutaneous and central-peritoneal adipose depots in three different conditions - lean, obese and obese diabetic - using immunohistochemistry. Our results suggest a correlation between the incidence of the stromal vascular cells and adipocytes TNF-α and TLR4 in the visceral depots in strong correlation with adipose tissue expansion. TLR2 positive cells were seen in the peripheral depots from all groups without any association with fat accumulation. These results focus on the existence of a new pathogenic pathway, the activation of TLR4, for the involvement of visceral adipose tissue in the activation and maintenance of the inflammatory cascade in obesity.

Hypoxia induced VEGF synthesis in visceral adipose depots of obese diabetic patients.

VEGF is one the pro-inflammatory adipokines synthesized by the "adipose secretoma" of obese subjects as a response to hypoxic conditions; but the main function of VEGF is angiogenesis, being recognized as the most important factor increasing blood capillaries in the adipose tissue by stimulating endothelial cell growth. In this paper, we propose a comparative study of the vascular response to VEGF synthesis in the subcutaneous and central-peritoneal adipose depots in lean, obese and obese diabetic patients. We used CD31 to label the endothelial cells in order to evaluate the response of the vascular network to VEGF synthesis. Our results showed an increase of VEGF protein synthesis in obese and obese-diabetic patients compared to lean subjects where the protein was absent. The positivity for VEGF in obese diabetic samples was observed in numerous structures from the adipose depots, both in the stromal vascular fraction--blood vessels and stromal cells--as well as in the cytoplasm of adipocytes. Positivity in the vascular wall was observed more frequently in areas of perivascular and intralobular fibrosis. Obese and diabetic patients showed similar incidence of CD31 immunoreactivity with lean subjects in both subcutaneous and peritoneal depots. In conclusion, human adipose depots show a different incidence of VEGF positive cells in relation with their disposal and the metabolic status. VEGF synthesis in visceral adipose tissue is inefficient being not followed by angiogenesis to counterbalance tissue hypoxia. We suggest that may be a pathogenic link between the degrees of intralobular fibrosis in adipose depots and VEGF expression.