MKK3/6-p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1 beta and TNF-alpha in immortalized periodontal ligament fibroblasts

Matrix metalloprotease-13 (MMP-13) or collagenase-3 is involved in a number of pathologic processes such as tumor metastasis and angiogenesis, osteoarthritis, rheumatoid arthritis and periodontal diseases. These conditions are associated with extensive degradation of both connective tissue and bone. This report examines gene regulation mechanisms and signal transduction pathways involved in Mmp-13 expression induced by proinflammatory cytokines in periodontal ligament (PDL) fibroblasts. Mmp-13 mRNA expression was increased 10.7 and 9.5 fold after stimulation with IL-1 beta (5 ng/mL) and TNF-alpha (10 ng/mL), respectively. However, inhibition of p38 MAPKinase with SB203580 resulted in significant (p < 0.001) induction (23.2 and 18.1 fold, respectively) of Mmp-13 mRNA as assessed by real time PCR. Negative regulation of IL-1 induced Mmp-13 expression was confirmed by inhibiting p38 MAPK gene expression with siRNA. Transient transfection of dominant negative forms of MKK3 and MKK6 also resulted in increased levels of Mmp-13 mRNA after IL-1 beta stimulation. Mmp-13 mRNA expression induced by TNF-alpha was decreased by JNK and ERK inhibition. Western blot and zymogram analysis indicated that Mmp-13 protein expression induced by the proinflammatory cytokines were also upregulated by inhibition of p38 MAPK. Reporter gene experiments using stable cell lines harboring 660-bp sequence of the murine Mmp-13 proximal promoter indicated that transcriptional mechanisms were at least partially involved in this negative regulation of Mmp-13 expression by p38 MAPK and upstream MKK3/6. These results suggest a negative transcriptional regulatory mechanism mediated by p38 MAPK and upstream MKK3/6 on Mmp-13 expression induced by proinflammatory cytokines in PDL fibroblasts. (c) 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Hypothalamic melanin-concentrating hormone is induced by cold exposure and participates in the control of energy expenditure in rats

Short-term cold exposure of homeothermic animals leads to higher thermogenesis and food consumption accompanied by weight loss. An analysis of cDNA-macroarray was employed to identify candidate mRNA species that encode proteins involved in thermogenic adaptation to cold. A cDNA-macroarray analysis, confirmed by RT-PCR, immunoblot, and RIA, revealed that the hypothalamic expression of melanin-concentrating hormone (MCH) is enhanced by exposure of rats to cold environment. The blockade of hypothalamic MCH expression by antisense MCH oligonucleotide in cold-exposed rats promoted no changes in feeding behavior and body temperature. However, MCH blockade led to a significant drop in body weight, which was accompanied by decreased liver glycogen, increased relative body fat, increased absolute and relative interscapular brown adipose tissue mass, increased uncoupling protein 1 expression in brown adipose tissue, and increased consumption of lean body mass. Thus, increased hypothalamic MCH expression in rats exposed to cold may participate in the process that allows for efficient use of energy for heat production during thermogenic adaptation to cold.

Decreased Total Antioxidant Capacity in Plasma, but Not Tissue, in Experimental Colitis

The aim of the present work was to compare colonic mucosa and plasmatic oxidative stress measured concomitantly and with different degrees of injury in rats with colitis induced by trinitrobenzene sulfonic acid. Three groups were studied: control group, colitis group, and colitis exacerbated by diclofenac. Enzymatic markers of colon injury showed enhanced activity in both groups with colitis. The colitis group treated with diclofenac presented higher colonic damage score than the other groups. In both groups with colitis, higher values of tert butyl hydroperoxide-initiated-chemiluminescence and thiobarbituric acid-reactive substances in tissue and decreased total radical-trapping antioxidant potential (TRAP) levels in plasma were found. In conclusion, independently of the degree of colonic mucosa injury and inflammation, oxidative stress in tissue occurs as a consequence of pro-oxidants increase, and is not explained by a reduction of antioxidant defenses. In both conditions, TRAP determination decreases in plasma, but not in tissue.

Comparative study of ovule and fruit development in species of Hypolytrum and Rhynchospora (Cyperaceae, Poales)

The development of the ovule and of the fruit of Hypolytrum bullatum and H. schraderianum (Mapanioideae) and of Rhynchospora consanguinea and R. rugosa (Cyperoideae) are described. All species share anatropous, bitegmic and crassinucellate ovules, funicular obturator, megagametophyte of the Polygonum type, presence of starch grains in the mature megagametophyte, free-nuclear endosperm, Onagrad-type embryogeny, testal-tegmic seed, and a simple fruit of the achene type. Rhynchospora species have characters typical of the family: micropyle formed by the inner integument alone; 3-4-layered parietal tissue; and hard achene. Hypolytrum species differ in those characters by presenting a slightly zigzag micropyle formed by both integuments connected with the funicular obturator, 5-8-layered parietal tissue, and fibrous-spongy achene. The peculiar formation of the micropyle in Hypolytrum is a feature reported here for the first time in the family. The ontogeny provides evidence for a better understanding of the dispersal unit in Hypolytrum supporting the classification as a true achene, like that of Rhynchospora, which is characteristic of the family.

Histopathological changes induced by extracts from the tissue covering the stingers of Potamotrygon falkneri freshwater stingrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proliferating cell nuclear antigen (PCNA) in non-Hodgkin's lymphomas: Correlation with working formulation and Kiel classification in formalin-fixed paraffin-embedded material

PCNA is a 36-KD proliferating cell nuclear antigen associated with the cell cycle. The immunocytochemical detection of PCNA represents a useful tool for the study of tumor proliferation activity. This study documents the detection of PCNA, using antibody PC 10 in formalin-fixed, paraffin-embedded tissue, and correlates the proliferative activity of the non-Hodgkin's lymphomas (NHL) with histological grading assessed by the International Working Formulation (WF) and Kiel classification. In 92 cases of NHLs we found a strong correlation between the PCNA index and lymphoma grading. Statistically significant differences were also found between the proliferative index (PI) in low and high grade lymphomas according to the Kiel classification (t = 9.519; p < 0.001) and between low, intermediate and high grade lymphomas according to the WF classification (F = 79.01; p < 0.001). In the Kiel classification the mean of low grade lymphomas was 39.5% and of high grade 75.7%. In the WF the average of low grade lymphomas was 29.7%, intermediate 53.1% and high 75.1%. Although the differences among the groups had been significant, we found variations inside each histological subgroup in both classifications. The intermediate lymphomas were the most heterogeneous group, with PI inside the same histologic subtypes coincident with low and high grade lymphomas. Since PCNA may be used as a marker of cell proliferation in clinical studies to estimate the biological aggressiveness of lymphomas, its determination in intermediate grade NHL could be very useful to evaluate individual cases in this group and determine prognosis and probably the appropriate therapy.

Reaction of rat connective tissue to implanted dentin tubes filled with a white mineral trioxide aggregate.

The purpose of this paper was to study the reaction of rat subcutaneous connective tissue to the implantation of dentin tubes filled with white mineral trioxide aggregate (MTA), a material that will be marketed. The tubes were implanted into rat subcutaneous tissue and the animals were sacrificed after 7 and 30 days. The undecalcified pieces were prepared for histological analysis with polarized light and von Kossa technique for mineralized tissues. Granulations birefringent to polarized light and an irregular structure like a bridge were observed next to the material; both were von Kossa positive. Also, in the dentin wall tubules a layer of birefringent granulations was observed. The results were similar to those reported for gray MTA, indicating that the mechanisms of action of the white and gray MTA are similar.

Cytokeratins in epithelia of odontogenic neoplasms

Neoplasms and tumours related to the odontogenic apparatus may be composed only of epithelial tissue or epithelial tissue associated with odontogenic ectomesenchyme. The immunohistochemical detection of different cytokeratins (CKs) polypeptides and vimentin has made it easier to explain the histogenesis of many epithelial diseases. The present study aimed to describe the immunohistochemical expression of cytokeratins 7, 8, 10, 13, 14, 18, 19 and vimentin in the epithelial components of the dental germ and of five types of odontogenic tumours. The results were compared and histogenesis discussed. All cells of the dental germ were positive for CK14, except for the preameloblasts and secreting ameloblasts, in which CK14 was gradually replaced by CK19. CK7 was especially expressed in the cells of the Hertwig root sheath and the stellate reticulum. The dental lamina was the only structure to express CK13. The reduced epithelium of the enamel organ contained CK14 and occasionally CK13. Cells similar to the stellate reticulum, present in the ameloblastoma and in the ameloblastic fibroma, were positive for CK13, which indicates a nature other than that of the stellate reticulum of the normal dental germ. The expression of CK14 and the ultrastructural aspects of the adenomatoid odontogenic tumour probably indicated its origin in the reduced dental epithelium. Calcifying odontogenic epithelial tumour is thought to be composed of primordial cells due to the expression of vimentin. Odontomas exhibited an immunohistochemical profile similar to that of the dental germ. In conclusion, the typical IF of odontogenic epithelium was CK14, while CK8, 10 and 18 were absent. Cytokeratins 13 and 19 labelled squamous differentiation or epithelial cells near the surface epithelium, and CK7 had variable expression.

Histological study of hamster buccal mucosa following topical application of DMBA and exposition to low power 337 nm laser light

The aim of the present work is to analyze the histological changes on hamster buccal mucosa caused by the topical use of 7,12-dimethylbenzanthracene (DMBA) and exposition to a 220 μJ/pulse nitrogen laser light (@ 337 nm) at an average power of 2,3 mW. Twenty-one hamsters divided into two experimental groups were treated six times with DMBA. One hamster was kept as control. Group I was composed by ten hamsters and was submitted only to DMBA. Group II, also with ten hamsters, received the same treatment as group I and was exposed to the laser radiation. The time duration of each irradiation section was 10 seconds. All the treatment happened in alternated days. The histological analysis took place twice, after the end of the treatment and after sixty days. Both experimental groups presented dilatation of vessels, thickening of the epithelial tissue and the presence of inflammatory infiltrates. The preliminary results indicates that in group II the number of dilated vessels and its new area are much more significant than in group I.

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system. © 2006 Coelho-Castelo et al; licensee BioMed Central Ltd.

Alkaline phosphatase localization in golden hamster epididymidis: A biochemical and histochemical investigation

The aim of this study was to determine alkaline phosphatase (ALP; E.C. 3.1.3.1) activity and major expression in homogenates obtained from different regions of Golden hamster epididymis, comprising the initial segment, head, body and tail, with concomitant research of this enzyme localization and activity in samples of tissues. These were collected from the same regions and investigated by histochemical conventional study performed on frozen histological sections. No significant differences in mean ALP activity, reported as U.100 mg-1 of tissue, were observed among the biological specimens collected from the epididymidis initial segment (0.92 ± 0.28 U.100 mg-1 tissue), head (1.07 ± 0.67 U.100 mg-1 tissue) and body (0.77 ± 0.23 U.100 mg-1 tissue). However, mean ALP activity was significantly higher in the epididymal tail (8.94 ± 0.40 U.100 mg-1 tissue) compared with the precedent segments. The findings suggested that ALP plays a significant role in the tail of the Golden hamster epididymidis, mediating androgenic segregation necessary to maintain the epithelial integrity. Furthermore, ALP acts on active transport of substances between the luminal fluid and spermatozoon membrane, and contrariwise. Thus, the high concentration of ALP in the epididymal tail helps to indicate the importance of this enzyme in the metabolism and maintenance of spermatozoa maturation and storage into the epididymidis luminal compartment, perhaps directly influencing the normal reproductive morphophysiology.

Clinical and histologic features of botryoid odontogenic cyst: A case report

Introduction. The lateral periodontal cyst, as the name implies, occurs on a lateral periodontal location and is of developmental origin, arising from cystic degeneration of clear cells of the dental lamina. A botryoid odontogenic cyst is considered to be a rare multilocular variant of a lateral periodontal cyst. Case presentation. We report the clinical and histopathologic features of a rare case of botryoid odontogenic cyst found in an edentulous area corresponding to the right lower canine of a 64-year-old African-American woman. A multilocular radiolucency was observed, and surgical removal of the lesion revealed a nodule of rubber-like consistency measuring about 1.5 cm in diameter. Cross-sectioning of the nodule showed that it consisted of various cystic compartments. Histologically, various voluminous periodic acid-Schiff-negative clear cells randomly distributed throughout the cystic epithelium were observed, as well as cell layers showing thickenings generally formed by oval, sometimes entangled plaques. The capsule consisted of fibrous connective tissue and showed rare and discrete foci of a perivascular mononuclear inflammatory infiltrate and reactive bone-tissue fragments. The final diagnosis was botryoid odontogenic cyst. Conclusion: We provide data that allow the reader to establish the differences between botryoid odontogenic cyst, glandular odontogenic cyst, and lateral periodontal cyst, helping with the differential diagnosis. The reader will have the opportunity to review botryoid odontogenic cyst clinical and histopathologic features, including treatment. © 2010 Farina et al; licensee BioMed Central Ltd.

Chronic Ethanol Consumption Alters All-Trans-Retinoic Acid Concentration and Expression of Their Receptors on the Prostate: A Possible Link Between Alcoholism and Prostate Damage

Background: Ethanol (EtOH) alters the all-trans-retinoic acid (ATRA) levels in some tissues. Retinol and ATRA are essential for cell proliferation, differentiation, and maintenance of prostate homeostasis. It has been suggested that disturbances in retinol/ATRA concentration as well as in the expression of retinoic acid receptors (RARs) contribute to benign prostate hyperplasia and prostate cancer. This study aimed to evaluate whether EtOH consumption is able to alter retinol and ATRA levels in the plasma and prostate tissue as well as the expression of RARs, cell proliferation, and apoptosis index. Methods: All animals were divided into 4 groups (n = 10/group). UChA: rats fed 10% (v/v) EtOH ad libitum; UChACo: EtOH-naïve rats without access to EtOH; UChB: rats fed 10% (v/v) EtOH ad libitum; UChBCo: EtOH-naïve rats without access to EtOH. Animals were euthanized by decapitation after 60 days of EtOH consumption for high-performance liquid chromatography and light microscopy analysis. Results: EtOH reduced plasma retinol concentration in both UChA and UChB groups, while the retinol concentration was not significantly different in prostate tissue. Conversely, plasma and prostate ATRA levels increased in UChB group compared with controls, beyond the up-regulation of RARβ and -γ in dorsal prostate lobe. Additionally, no alteration was found in cell proliferation and apoptosis index involving dorsal and lateral prostate lobe. Conclusions: We conclude that EtOH alters the plasma retinol concentrations proportionally to the amount of EtOH consumed. Moreover, high EtOH consumption increases the concentration of ATRA in plasma/prostate tissue and especially induces the RARβ and RARγ in the dorsal prostate lobe. EtOH consumption and increased ATRA levels were not associated with cell proliferation and apoptosis in the prostate. © 2012 by the Research Society on Alcoholism.

Healing outcomes at implants installed in sites augmented with particulate autologous bone and xenografts. An experimental study in dogs

Aim: To evaluate the integration of implants installed at the interface of pristine and grafted tissue augmented with particulate autologous bone or deproteinized bovine bone mineral (DBBM), concomitantly with a collagen membrane. Material and methods: In 6 Labrador dogs, the distal root of 3P3 and 4P4 was endodontically treated and hemi-sected, and the mesial roots extracted concomitantly with the extraction of 2P2. The buccal bony walls were removed, and two box-shaped defects, one larger and one smaller, were created. After 3 months, flaps were elevated, and the defects were filled with particulate autologous bone or DBBM in the right and left side of the mandible, respectively. Collagen membranes were used to cover the grafted areas. Three months later, flaps were elevated, and a customized device was used as surgical guide to prepare the recipient sites at the interface between grafts and pristine bone. One implant was installed in each of the four defects. After 3 months, biopsies were harvested and ground sections prepared for histological evaluation. Results: The augmentation technique was effective at all sites and all the foreseen implants were installed. In the histological analysis, all implants were integrated in mature bone, at both the buccal and lingual aspects. The most coronal bone-to-implant contact and the top of the buccal bony crest were located at a similar distance between test and control implants. However, these distances were higher at the larger compared with the smaller defects. Especially in the large defect, residual particles of DBBM were found embedded into connective tissue and located outside the bony crest. Conclusions: Particulate autologous bone as well as DBBM particles used to augment horizontally the alveolar bony process allowed for the osseointegration of implants installed after 3 months of healing. © 2012 John Wiley & Sons A/S.

Influence of buccal bony crest width on marginal dimensions of peri-implant hard and soft tissues after implant installation. An experimental study in dogs

Aim: To evaluate the influence of the width of the buccal bony wall on hard and soft tissue dimensions following implant installation. Material and methods: Mandibular premolars and first molars of six Labrador dogs were extracted bilaterally. After 3 months of healing, two recipient sites, one on each side of the mandible, were prepared in such a way as to obtain a buccal bony ridge width of about 2 mm in the right (control) and 1 mm in the left sides (test), respectively. Implants were installed with the coronal margin flush with the buccal alveolar bony crest. Abutments were placed and the flaps were sutured to allow a non-submerged healing. After 3 months, the animals were euthanized and ground sections obtained. Results: All implants were completely osseointegrated. In respect to the coronal rough margin of the implant, the most coronal bone-to-implant contact was apically located 1.04 ± 0.91 and 0.94 ± 0.87 mm at the test and control sites, respectively, whereas the top of the bony crest was located 0.30 ± 0.40 mm at the test and 0.57 ± 0.49 mm at the control sites. No statistically significant differences were found. A larger horizontal bone resorption, however, evaluated 1 mm apically to the rough margin, was found at the control (1.1 ± 0.7 mm) compared to the test (0.3 ± 0.3 mm) sites, the difference being statistically significant. A thin peri-implant mucosa (2.4-2.6 mm) was found at implant installation while, after 3 months of healing, a biological width of 3.90-4.40 mm was observed with no statistically significant differences between control and test sites. Conclusions: A width of the buccal bony wall of 1or 2 mm at implant sites yielded similar results after 3 months of healing in relation of hard tissue and soft tissues dimensions after implant installation. © 2012 John Wiley & Sons A/S.