Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-1 beta, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1 beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TCF-beta1 and IL-1 beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TCF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the nation of distinct functions for these cell-surface proteoglycans. (C) 2001 Wiley-Liss, inc.

The effects of copper on the microbial community of a coral reef sponge

Marine sponges often harbour communities of symbiotic microorganisms that fulfil necessary functions for the well-being of their hosts. Microbial communities associated with the sponge Rhopaloeides odorabile were used as bioindicators far sublethal cupric ion (Cu2+) stress. A combined strategy incorporating molecular, cultivation and electron microscopy techniques was adopted to monitor changes in microbial diversity. The total density of sponge-associated bacteria and counts of the predominant cultivated symbiont (alpha -proteobacterium strain NW001) were significantly reduced in response to Cu2+ concentrations of 1.7 mug l(-1) and above after 14 days of exposure. The number of operational taxonomic units (OTUs) detected by restriction fragment length polymorphism (RFLP) decreased by 64% in sponges exposed to 223 mug l(-1) Cu2+ for 48 h and by 46% in sponges exposed to 19.4 mug l(-1) Cu2+ for 14 days. Electron microscopy was used to identify 17 predominant bacterial morphotypes, composing 47% of the total observed cells in control sponges. A reduction In the proportion of these morphotypes to 25% of observed cells was evident in sponges exposed to a Cu2+ concentration of 19.4 mug l(-1). Although the abundance of most morphotypes decreased under Cu2+ stress, three morphotypes were not reduced in numbers and a single morphotype actually increased in abundance. Bacterial numbers, as detected using fluorescence in situ hybridization (FISH), decreased significantly after 48 h exposure to 19.4 mug l(-1) Cu2+. Archaea, which are normally prolific in R. odorabile, were not detected after exposure to a Cu2+ concentration of 19.4 mug l(-1) for 14 days, indicating that many of the microorganisms associated with R. odorabile are sensitive to free copper. Sponges exposed to a Cu2+ concentration of 223 mug l(-1) became highly necrosed after 48 h and accumulated 142 +/- 18 mg kg(-1) copper, whereas sponges exposed to 19.4 mug l(-1) Cu2+ accumulated 306 +/- 15 mg kg(-1) copper after 14 days without apoptosis or mortality. Not only do sponges have potential for monitoring elevated concentrations of heavy metals but also examining changes in their microbial symbionts is a novel and sensitive bioindicator for the assessment of pollution on important microbial communities.

MMP-9 and CD68(+) cells are required for tissue remodeling in response to natural hydroxyapatite

Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. The aim of this study was to examine, through immunohistochemistry approach, the involvement of MMP-9 and CD68(+) cells during tissue remodeling in response to natural hydroxyapatite (HA) implanted in rat subcutaneous tissue. Before experimentation, forty animals were randomly distributed into two experimental groups: Group-I (Gen-Ox (TM) micro-granules) and Group-II (Gen-Ox (TM) macro-granules). Afterwards, the biopsies were collected after 10, 20, 30, and 60 days post-implantation. Our results showed that at 10 days, a low-renewal foreign body type granuloma formation was observed in most of the cases. Macrophage- and fibroblast-like cells were the predominant type of cells positively stained for MMP-9 in both groups. Once macrophage-like cells seemed to be the major source of MMP9, antibody against pan-CD68 epitope was used to correlate these findings. In agreement, MMP-9 and CD68(+) cells were distributed at the periphery and the central region of the granuloma in all experimental periods, however no staining was observed in cell contacting to material. Besides macrophages, the lysosomal glycoprotein epitope recognized by CD68 antibodies can be expressed by mast cell granules and sometimes by fibroblasts. Taken together, our results suggest that xenogenic HA promotes extracellular matrix remodeling through induction of MMP-9 activity and presence of CD68(+) cells.

Cytotoxic effects and pulpal response caused by a mineral trioxide aggregate formulation and calcium hydroxide

Purpose: To evaluate: the in vivo pulpal response after pulpotomy with different capping agents. In addition, the in vitro cytotoxic effects of both materials were assessed by applying them on culture of pulp cells. Methods: For the in vivo test, the coronal pulp of 28 teeth of dogs was mechanically removed and the root pulps were capped with the following dental materials: Group 1: Pro-Root NITA (PRMTA); and Group 2 (control): calcium hydroxide saline paste (CH). After 60 days, the animals were sacrificed and the teeth processed for histological analysis. In the in vitro test, experimental extracts obtained from both capping agents were applied on the cultured MDPC-23 odontoblast-like cells. Results: In the root pulps capped with PRMTA or CH, coagulation necrosis partially replaced by dystrophic calcification as well as tubular dentin matrix laid down by elongated pulp cells was observed. None or mild inflammatory response occurred beneath the capped pulpal wound. Regarding the pulpal response, PRMTA and CH presented no statistical difference. However, the teeth capped CH presented greater healthy pulp loss which resulted in convex shape of the hard barrier than PRMTA. When applied on the cultured cells, it was demonstrated that PRMTA and CH solutions decreased the cell metabolic activity by 9.9% and 29.4%, respectively. CH caused higher cytotoxic effects to the MDPC-23 cells as well as deeper healthy pulp tissue loss than PRMTA. However, similar sequence of healing occurred after pulpotomy with both dental materials.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.

Transcriptional response of murine macrophages upon infection with opsonized Paracoccidioides brasiliensis yeast cells

Paracoccidioides brasiliensis is the etiologic agent of the Paracoccidioidomycosis the most common systemic mycosis in Latin America. Little is known about the regulation of genes involved in the innate immune host response to P. brasiliensis. We therefore examined the kinetic profile of gene expression of peritoneal macrophage infected with P. brasiliensis. Total RNA from macrophages at 6, 24 and 48 h was extracted, hybridized onto nylon membranes and analyzed. An increase in the transcription of a number of pro-inflammatory molecules encoding membrane proteins, metalloproteases, involved in adhesion and phagocytosis, are described. We observed also the differential expression of genes whose products may cause apoptotic events induced at 24 h. In addition, considering the simultaneous analyses of differential gene expression for the pathogen reported before by our group, at six hours post infection, we propose a model at molecular level for the P. brasiliensis-macrophage early interaction. In this regard, P. brasiliensis regulates genes specially related to stress and macrophages, at the same time point, up-regulate genes related to inflammation and phagocytosis, probably as an effort to counteract infection by the fungus. (c) 2007 Elsevier Masson SAS. All fights reserved.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Modulatory role of locus coeruleus and estradiol on the stress response of female rats

The activity of the hypothalamic-pituitary-adrenal axis is modulated by the norepinephrinergic system and, in females, also by the ovarian hormones. We investigated the role of ovarian steroids and the locus coeruleus (LC) on stress-induced corticosterone secretion in female rats. Ovariectomized rats without hormonal replacement (OVX) or treated with estradiol (OVE) or estradiol plus progesterone (OVEP) were subjected to jugular cannulation. Immediately after that, each hormonal treatment group was subjected to LC lesion or sham surgery or no brain surgery. After 24 h, blood samples of all 9 groups were collected before and after ether inhalation. Other four groups (OVX control, sham and lesioned, and OVE) were perfused for glucocorticoid receptor (GR) immunocytochemistry in hippocampal CA1 neurons and paraventricular nucleus (PVN). Estradiol replacement decreased while LC lesions increased stress-induced corticosterone secretion. The effect of LC lesion was potentiated with the removal of ovarian steroids. Since GR expression of lesioned animals decreased in the hippocampus, but not in PVN, we suggest that the effect of LC lesion on corticosterone secretion could be due to a reduction in the efficiency of the negative feedback system in the CA1 neurons. However, this mechanism is not involved in the estradiol modulation on corticosteroid secretion, as no change in GR expression was observed in estradiol-treated animals.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009

Locus coeruleus mediates cold stress-induced polycystic ovary in rats

Previous reports about the rat ovary have shown that cold stress promotes ovarian morphological alterations related to a polycystic ovary (PCO) condition through activation of the ovarian sympathetic nerves. Because the noradrenergic nucleus locus coeruleus (LC) is activated by cold stress and synaptically connected to the preganglionic cell bodies of the ovarian sympathetic pathway, this study aimed to evaluate the LC`s role in cold stress-induced PCO in rats. Ovarian morphology and endocrine and sympathetic functions were evaluated after 8 wk of chronic intermittent cold stress (4 C, 3 h/d) in rats with or without LC lesion. The effect of acute and chronic cold stress upon the LC neuron activity was confirmed by Fos protein expression in tyrosine hydroxylase-immunoreactive neurons. Cold stress induced the formation of follicular cysts, type III follicles, and follicles with hyperthecosis alongside increased plasma estradiol and testosterone levels, irregular estrous cyclicity, and reduced ovulation. Considering estradiol release in vitro, cold stress potentiated the ovarian response to human chorionic gonadotropin. Ovarian norepinephrine (NE) was not altered after 8 wk of stress. However, LC lesion reduced NE activity in the ovary of cold-stressed rats, but not in controls, and prevented all the cold stress effects evaluated. Cold stress increased the number of Fos/tyrosine hydroxylase-immunoreactive neurons in the LC, but this effect was more pronounced for acute stress as compared with chronic stress. These results show that cold stress promotes PCO in rats, which apparently depends on ovarian NE activity that, under this condition, is regulated by the noradrenergic nucleus LC.

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint. (C) 2007 Elsevier Inc. All rights reserved.

Temporal relationship between the auditory brainstem response and focal responses of auditory nerve root and cochlear nucleus during development in the tammar wallaby (Macropus eugenii)

Thirty-two pouch-young tammar wallabies were used to discover the generators of the auditory brainstem response (ABR) during development by the use of simultaneous ABR and focal brainstem recordings. A click response from the auditory nerve root (ANR) in the wallaby was recorded from postnatal day (PND) 101, when no central auditory station was functional, and coincided with the ABR, a simple positive wave. The response of the cochlear nucleus (CN) was detected from PND 110, when the ABR had developed 1 positive and 1 negative peak. The dominant component of the focal ANR response, the N-1 wave, coincided with the first half of the ABR P wave, and that of the focal CN response, the N-1 wave, coincided with the later two thirds. In older animals, the ANR response coincided with the ABR's N-1, wave, while the CN response coincided with the ABR's P-2, N-2 and P-3 waves, with its contribution to the ABR P-2 dominant. The protracted development of the marsupial auditory system which facilitated these correlations makes the tammar wallaby a particularly suitable model. Copyright (C) 2001 S. Karger AG, Basel.

Increasing the response rate to a mailed questionnaire by including more stamps on the return envelope: A cotwin control study

Twins taking part in two unrelated studies were sent a questionnaire together with a self-addressed envelope that either carried one or multiple (up to 5) stamps to the same value. The unprompted proportion of questionnaires returned (before commencement of telephone reminder calls) was increased from 62% to 71% in one study, and from 43% to 52% in the other study (test for common odds ratio in studies, p = 0.04).

Saturable dose-response relationships for melphalan in melanoma treatment by isolated limb infusion in the nude rat

Nude rats bearing melanomas on their hindlimbs were treated by isolated limb infusion (ILI) with increasing doses (7.5-400 mug/ml) of melphalan. The response of tumours to treatment at the end of the observation period was graded, according to diameter, as complete response (CR), partial response (PR), no change (NC) or progressive disease (PD). No linear relationship between the dose of melphalan and the tumour response was observed. All doses above a threshold of 15 mug/ml achieved a PR or CR. The achievement of CR was not related to increased dose. Two major implications arise from this work. Firstly, the typically two-to three-fold increase in cytotoxic drug concentration given in high dose chemotherapy compared with standard drug concentration may not be sufficient to produce the expected increase in tumour response and possibly survival, and the controversial results of high dose chemotherapy in different studies may thus be explained. Secondly, since an increase in melphalan dose above a certain threshold does not greatly increase tumour response, the use of combination therapies would seem to be more likely to be effective than increased chemotherapeutic drug doses in achieving better tumour responses.