Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.     

Immunohistochemical characterization of elastic system fibers in rat molar periodontal ligament.

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.     

Increased local matrix metalloproteinase-8 expression in the periodontal connective tissues of smokers with periodontal disease

Matrix metalloproteinase (MMP)-8 has been associated with the progression of periodontitis, a common inflammatory disease of the supporting structures of the teeth, and with other degradative diseases. Tobacco smokers are at high risk of developing periodontitis that may progress more rapidly and respond poorly to treatment. Therefore, MMP-8 expression was determined by immunofluorescence staining in 60 random, computer-selected fields in the excised periodontal tissues of smokers and non-smokers, balanced for age, gender, and periodontal status. Immunofluorescence intensity, representing MMP-8 expression, in the periodontal tissues of smokers (30 fields from 6 subjects, mean 1154+/-124 units) was significantly higher than that in the periodontal tissues of non-smokers (30 fields from 6 subjects, mean 817+/-60 units; p < 0.05). Serum MMP-8 concentrations were measured by ELISA and compared in a larger group of smokers (n = 20) and age- and gender-balanced non-smokers (n = 20). Systemic MMP-8 concentrations in smokers and non-smokers were not significantly different (p > 0.05). A local tobacco-related increase in MMP-8 burden may contribute to periodontal disease progression in tobacco smokers. This finding may also have relevance to other tobacco-induced inflammatory diseases, such as vascular and pulmonary diseases.     

cav-p60 expression in rat muscle tissues

Caveolae are plasmalemmal invaginations of uncertain function. In view of the large number of hypotheses on caveolar functions, it is important to identify which components of caveolae are tissue specific and which are general. The only well-characterized major protein of caveolae is caveolin, which exists in three tissue-specific isoforms: caveolin-1, -2, and -3. Recently cav-p60 was characterized as a 60-kDa caveola-specific protein in adipocytes. The distributions of cav-p60 and caveolin isoforms in different rat muscle tissues were examined by immunofluorescence and immunoelectron microscopy. Cav-p60 was present in caveolae of skeletal and heart muscle, in vascular and intestinal smooth muscle, and in adipocyte caveolae. Furthermore cav-p60 was present in endothelial cells and cells of perineural sheaths. Caveolin-1 and -2 were present in adipocytes, endothelial cells, and cells of perineural sheaths. In all kinds of vascular and intestinal smooth muscle, caveolin-1 and -2 were present at high levels, whereas caveolin-3 expression was low or undetectable, depending on the specific smooth muscle subtype. High levels of caveolin-3 were found only in caveolae and T tubules of skeletal and heart muscle. We conclude that cav-p60 is a highly specific marker of caveolae in many if not all cell types having caveolae.     

Plasminogen activator inhibitor type 1 expression induced by lipopolysaccharide of Porphyromonas gingivalis in human gingival fibroblast

In the gingival tissues of patients with periodontitis, inflammatory responses are mediated by a wide variety of genes. In our previous screening study, plasminogen activator inhibitor type 1 (PAI-1) mRNA binding protein expression was increased in gingiva from periodontitis patients. In this study, we further investigated the signaling pathway involved in PAI-1 expression induced by Porphyromonas gingivalis LPS (Pg LPS) in human gingival fibroblasts (HGF). When HGFs were treated with Pg LPS, both PAI-1 mRNA expression and PAI-1 protein were induced in a dose-dependent manner. Pg LPS induced NF-κB activation and the expressions of PAI-1 mRNA and protein were suppressed by pretreating with a NF-κB inhibitor. Pg LPS also induced ERK, p38, and JNK activation, and Pg LPS-induced PAI-1 expression was inhibited by ERK/p38/JNK inhibitor pretreatment. In conclusion, Pg LPS induced PAI-1 expression through NF-κB and MAP kinases activation in HGF.     

Estradiol distribution and penetration in rat skin after topical application, studied by high resolution autoradiography

Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography. 3H-estradiol-17 beta was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm2 to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol 3H-estradiol x cm-2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm2, a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicuous in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.     

Calcitonin gene expression induced by lipopolysaccharide in the rat pituitary

Procalcitonin (PCT), the precursor protein of calcitonin (CT), has been considered recently as a significant indicator of bacterial infection and sepsis. However, the major source of PCT in sepsis remains unclear. The hypothalamic-pituitary-adrenal axis is activated during sepsis. Moreover, immunoreactive CT (iCT) can be detected in the pituitary. Therefore, we examined the effects of lipopolysaccharide (LPS) administration on CT mRNA expression in the pituitary. After administration of LPS, CT mRNA expression in the pituitary was increased significantly. The increase of CT mRNA was associated with significant elevations of the iCT levels in the serum. These results imply that the pituitary is one of the sources of the serum PCT during sepsis.     

Estradiol distribution and penetration in rat skin after topical application,studied by high resolution autoradiography

Summary Transdermal pathways and targets in the skin for estradiol were investigated using dry-mount autoradiography.³H-estradiol-17 was applied at doses of 30.1 pmol, 120.4 pmol and 301 pmol/cm² to shaved rat skin in the dorsal neck region. Vehicles were DMSO, ethylene glycol or sesame oil. After 2 h of topical treatment with 30.1 pmol³H-estradiol×cm^–2 dissolved in DMSO a distinct cellular distribution was apparent. Target cells with concentrations of radioactivity were found in epidermis, sebaceous glands, dermal papillae of hair and fibroblasts. After treatment with 120.4 and 301 pmol/cm², a penetration gradient of radioactivity was recognizable however it masked specific cellular and subcellular uptake. The stratum corneum accumulated and retained radioactivity, apparently forming a depot for the hormone. Strong concentration and retention of the hormone was conspicious in sebaceous glands for more than 24 h, suggesting that sebaceous glands serve as a second storage site for the hormone. In all autoradiograms two penetration pathways to the dermis were visible: one through the stratum corneum and epidermis, the other through the hair canals and hair sheaths.This work was supported by US PHS grant NS09914     

Cyclooxygenase-1 and cyclooxygenase-2 expression in rat kidney and adrenal gland after stimulation with systemic lipopolysaccharide

Cyclooxygenase-2 (COX-2) is a recently discovered isoform of cyclooxygenase that is inducible by various types of inflammatory stimuli. Although this enzyme is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins, the precise location, distribution, and regulation of prostaglandin synthesis remains unclear in several tissues. Using in situ hybridization histochemistry, we investigated the induction of COX-1 and COX-2 mRNA expression after systemic administration of a pyrogen, lipopolysaccharide (LPS), in kidney and adrenal gland in the rat. The COX-2 mRNA signals dramatically increased 1 h after LPS treatment in the kidney outer medulla and adrenal cortex, where almost no or little expression was observed in nontreated animals, and returned to control levels within 24 h. COX-2 mRNA levels increased in the kidney inner medulla 6 h after treatment. There was also a significant increase in mRNA levels in the kidney cortex and adrenal medulla. On the other hand, COX-1 mRNA levels did not show any detectable changes except in the kidney inner medulla, where a significant downregulation of mRNA expression was observed after LPS treatment. Light and electron immunocytochemistry using COX-2 antibodies showed that strong COX-2 immunoreactivity was localized to certain cortical cells of the thick ascending limb of Henle. In addition, based on double-staining with antiserum to nitric oxide synthase (NOS) four further cell populations could be identified in kidney cortex, including weakly COX-2-positive, NOS-positive macula densa cells. After LPS treatment, changes in COX-2 immunoreactivity could be observed in interstitial cells in the kidney medulla and in inner cortical cells in the adrenal gland. These results show that COX-2 is a highly induced enzyme that can be up-regulated in specific cell populations in kidney and adrenal gland in response to inflammation, leading to the elevated levels of prostaglandins seen during fever. In contrast COX-1 mRNA levels remained unchanged in this experimental situation, except for a decrease in kidney inner medulla.     

Vasohibin-1 promotes osteoclast differentiation in periodontal disease by stimulating the expression of RANKL in gingival fibroblasts

Vasohibin-1 (VASH1) is a key inhibitor of vascular endothelial growth factor-induced angiogenesis. Although the involvement of VASH1 in various pathological processes has been extensively studied, its role in periodontal disease (PD) remains unclear. We aimed to investigate the role of VASH1 in PD by focusing on osteoclastogenesis regulation. We investigated VASH1 expression in PD by analyzing data from the online Gene Expression Omnibus (GEO) database and using a mouse ligature-induced periodontitis model. The effects of VASH1 on osteoclast differentiation and osteoclastogenesis-supporting cells were assessed in mouse bone marrow-derived macrophages (BMMs) and human gingival fibroblasts (GFs). To identify the stimulant of VASH1, we used culture broth from Porphyromonas gingivalis (Pg), a periopathogen. The GEO database and mouse periodontitis model revealed that VASH1 expression was upregulated in periodontitis-affected gingival tissues, which was further supported by immunohistochemistry and qRT-PCR analyses. VASH1 expression was significantly stimulated in GFs after treatment with the Pg broth. Direct treatment with recombinant VASH1 protein did not stimulate osteoclast differentiation in BMMs but did contribute to osteoclast differentiation by inducing RANKL expression in GFs through a paracrine mechanism. Small interfering RNA-mediated silencing of VASH1 in GFs abrogated RANKL-mediated osteoclast differentiation in BMMs. Additionally, VASH1-activated RANKL expression in GFs was significantly suppressed by MK-2206, a selective inhibitor of AKT. These results suggest that Pg-induced VASH1 may be associated with RANKL expression in GFs in a paracrine manner, contributing to osteoclastogenesis via an AKT-dependent mechanism during PD progression.     

4-Hydroxynonenal modifies IgA in rat intestine after lipopolysaccharide injection

The lipid peroxide 4-hydroxynonenal (HNE) was measured in rat intestinal mucosa after lipopolysaccharide (LPS) injection (0.5 mg/kg, ip) by a highly sensitive time-resolved fluoroimmunoassay. HNE was increased, with a small peak at 20 min followed by a sustained elevation at 2-4 h, after injection of LPS. Immunohistochemistry demonstrated enhanced labeling with anti-IgA and anti-HNE antibodies in the plasma cells followed by diffusion of the labeled materials into the submucosal tissue after LPS injection. Immunoprecipitation with anti-IgA antibody and Western blotting with anti-HNE antibody showed that IgA is modified with HNE after LPS injection. The HNE (5 microM-5 mM) modification in vitro reduced the bactericidal activity of IgA and anti-Escherichia coli serum. The HNE modification in vitro also promoted polymerization of IgA as shown by nondenaturing gel electrophoresis. This is the first demonstration of the modification of IgA with HNE in an in vivo model of intestinal inflammation as well as in vitro effects of HNE on bactericidal activity and polymerization of IgA. These findings will help in understanding the involvement of oxidative stress in the IgA-mediated immune response exerted by plasma cells in early intestinal inflammation.     

Upregulation of CBLL1 in rat brain cortex after lipopolysaccharide treated

CBLL1 (Casitas B-lineage lymphoma-transforming sequence-like protein 1) also known as Hakai, was originally identified as an E3 ubiquitin-ligase for the E-cadherin complex. Recent data have provided evidences for novel biological functional role of CBLL1 during tumor progression and other diseases. However, its distribution and function in the central nervous system (CNS) remains unclear. In this study, we found CBLL1 was significant up-regulation in cerebral cortex after LPS administration and immunofluorescent labeling indicated that CBLL1 was localized striking in the neurons. We also investigated co-staining of CBLL1 and active-caspase-3 and cyclin D1 in the cerebral cortex following LPS administration. Based on our data, we speculated that CBLL1 might play an important role in neuronal apoptosis following LPS administration and might provide a basis for the further study on its role in cell cycle re-entry in neuroinflammation in CNS.     

人牙龈成纤维细胞与牙周膜细胞的生物活性

采用组织块法分离培养牙周膜细胞和牙龈成纤维细胞,测定二者的增殖特性和ALP活性,利用免疫组化和FCM方法比较Ⅰ、Ⅲ型胶原、BMP的表达情况,以观察对比两种细胞的生物学特性的异同。找出牙龈成纤维细胞和牙周膜细胞在胶原基质合成方面存在差异,发现Ⅰ、Ⅲ型胶原可作为鉴别两种细胞的标志物,ALP与BMP可作为鉴别两种细胞的标志,牙周膜细胞比牙龈成纤维细胞具有较强的成骨能力。从而为今后改良两种细胞成为牙周组织工程的种子细胞奠定基础。     

Cytokine profiles in mice tissues after irradiation of the thymus projection area with femtosecond laser

The effects of pulsed femtosecond laser irradiation in the near ultraviolet region on the levels of cytokines in the thymus, blood, and skin of irradiated mice have been studied. Irradiation of the thymus projection area with low-intensity laser radiation in the near UV region of the spectrum showed significant changes in cytokine levels in the skin and thymus and, to a lesser extent, in the blood of irradiated mice. Laser irradiation with a power density of 20 mW/cm² affects the cytokine profile in the thymus: IFN-γ, IL-3, IL-4, IL-5, eotaxin, GM-CSF, and chemokine KC–factors that can affect differentiation and proliferation of the cells of the immune system in the gland. Conclusions: It is assumed that changes in the expression of cytokines in the thymus after laser irradiation are explained by the rearrangement of biochemical processes possibly associated with the maturation of cells in the gland.     

Cytokine gene expression in the islets of the diabetic Biobreeding/Worcester rat

The Biobreeding Worcester rat provides one of the best models of autoimmune diabetes. Immunopathologic studies of acute diabetes show that the islets are infiltrated by T cells and macrophages. It has been hypothesized that the islets are damaged by the secretion of cytokines such as IL-1 and TNF-alpha and that their function may be altered by IL-6. In this study, we utilized in situ hybridization to determine the expression of the IL-1, TNF, and IL-6 genes within the pancreas of the acute diabetic Biobreeding Worcester rat. These studies showed that cells expressing IL-1, TNF, and IL-6 were present within the islets and in the exocrine pancreas surrounding islets, ducts, and vessels and in an interstitial location. Cells expressing TNF and IL-1 mRNA were present in about 20% of the islets, whereas cells expressing IL-6 were present in about 4% of the islets. Islets containing TNF- or IL-1-positive cells contained about three positive cells per islet whereas only about one IL-6-positive cell was present per islet. In 26% of the islets peri-insular TNF-positive cells were found. Peri-insular IL-1 positive cells were seen in 14% of the islets and 8% showed peri-insular IL-6 positive cells. In nondiabetic 30-day old DP or 90-day-old DR rats intra-islet cytokine gene expression was not seen. Our studies support the view that cytokines are important in beta cell destruction.     

Cardiovascular effects of mycotoxin T-2 after topical application in rats

Evidence has been mounting that trichothecenes cause cardiac lesions and cardiovascular effects in general. T-2 toxin, dissolved in dimethyl sulfoxide, was applied in doses of 0, 1.0, 2.0 mg/kg to the skin of Sprague-Dawley rats. Twenty-four hours later, the cardiac function of the animals was assessed, followed by killing and histological examination. It was found that the arterial blood pressure values were lower in the 2.0 mg/kg group, the peak intraventricular pressure was lower in both the 1.0 and 2.0 mg/kg groups, and the resting systolic and diastolic blood pressure values of the 2.0 mg/kg group were lower than the 0 and 1.0 mg/kg groups. The 1.0 and 2.0 mg/kg groups had significantly lower epinephrine-stimulated intraventricular pressure values, indicating reduced contractility. Extended Q-T intervals in electrocardiograms of the 1.0 and 2.0 mg/kg groups suggested also impaired contractility. The histological examination gave equivocal results. It is concluded that topical applications of small doses of T-2 toxin have a noticeable negative effect on cardiovascular function.