Clinical and histologic evaluation of granular Beta-tricalcium phosphate for the treatment of human intrabony periodontal defects: a report on five cases

BACKGROUND: The aim of the study is to clinically and histologically evaluate the healing of advanced intrabony defects treated with open flap debridement and the adjunct implantation of granular beta tricalcium phosphate (beta-TCP). METHODS: Five patients, each displaying advanced combined 1- and 2-wall intrabony defects around teeth scheduled for extraction or root resection, were recruited. Approximately 6 months after surgery, the teeth or roots were removed together with a portion of their surrounding soft and hard tissues and processed for histologic evaluation. RESULTS: The mean probing depth (PD) was reduced from 10.8 +/- 2.3 mm presurgically to 4.6 +/- 2.1 mm, whereas a mean clinical attachment level (CAL) gain of 5.0 +/- 0.7 mm was observed. The increase in gingival recession was 1.2 +/- 3.2 mm. The histologic evaluation indicated the formation of new cellular cementum with inserting collagen fibers to a varying extent (mean: 1.9 +/- 0.7 mm; range: 1.2 to 3.03 mm) coronal to the most apical extent of the root instrumentation. The mean new bone formation was 1.0 +/- 0.7 mm (range: 0.0 to 1.9 mm). In most specimens, beta-TCP particles were embedded in the connective tissue, whereas the formation of a mineralized bone-like or cementum-like tissue around the particles was only occasionally observed. CONCLUSION: The present data indicates that treatment of intrabony periodontal defects with this beta-TCP may result in substantial clinical improvements such as PD reduction and CAL gain, but this beta-TCP does not seem to enhance the regeneration of cementum, periodontal ligament, and bone.

Expression of adipokines and estrogen receptors in adipose tissue and placenta of patients with gestational diabetes mellitus

The purpose of this study was to assess the expression profile of genes with potential role in the development of insulin resistance (adipokines, cytokines/chemokines, estrogen receptors) in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placenta of pregnant women with gestational diabetes mellitus (GDM) and age-matched women with physiological pregnancy at the time of Caesarean section. qRT-PCR was used for expression analysis of the studied genes. Leptin gene expression in VAT of GDM group was significantly higher relative to control group. Gene expressions of interleukin-6 and interleukin-8 were significantly increased, whereas the expressions of genes for estrogen receptors alpha and beta were significantly reduced in SAT of GDM group relative to controls, respectively. We found no significant differences in the expression of any genes of interest (LEP, RETN, ADIPOR1, ADIPOR2, TNF-alpha, CD68, IL-6, IL-8, ER alpha, ER beta) in placentas of women with GDM relative to controls. We conclude that increased expression of leptin in visceral adipose depot together with increased expressions of proinflammatory cytokines and reduced expressions of estrogen receptors in subcutaneous fat may play a role in the etiopathogenesis of GDM.

Cis-4-methylsphingosine is a sphingosine-1-phosphate receptor modulator

Sphingosine-1-phosphate (S1P) acts as high affinity agonist at specific G-protein-coupled receptors, S1P(1-5), that play important roles e.g. in the cardiovascular and immune systems. A S1P receptor modulating drug, FTY720 (fingolimod), has been effective in phase III clinical trials for multiple sclerosis. FTY720 is a sphingosine analogue and prodrug of FTY720-phosphate, which activates all S1P receptors except S1P(2) and disrupts lymphocyte trafficking by internalizing the S1P(1) receptor. Cis-4-methylsphingosine (cis-4M-Sph) is another synthetic sphingosine analogue that is readily taken up by cells and phosphorylated to cis-4-methylsphingosine-1-phosphate (cis-4M-S1P). Therefore, we analysed whether cis-4M-Sph interacted with S1P receptors through its metabolite cis-4M-S1P in a manner similar to FTY720. Indeed, cis-4M-Sph caused an internalization of S1P receptors, but differed from FTY720 as it acted on S1P(2) and S1P(3) and only weakly on S1P(1), while FTY720 internalized S1P(1) and S1P(3) but not S1P(2). Consequently, pre-incubation with cis-4M-Sph specifically desensitized S1P-induced [Ca(2+)](i) increases, which are mediated by S1P(2) and S1P(3), in a time- and concentration-dependent manner. This effect was not shared by sphingosine or FTY720, indicating that metabolic stability and targeting of S1P(2) receptors were important. The desensitization of S1P-induced [Ca(2+)](i) increases was dependent on the expression of SphKs, predominantly of SphK2, and thus mediated by cis-4M-S1P. In agreement, cis-4M-S1P was detected in the supernatants of cells exposed to cis-4M-Sph. It is concluded that cis-4M-Sph, through its metabolite cis-4M-S1P, acts as a S1P receptor modulator and causes S1P receptor internalization and desensitization. The data furthermore help to define requirements for sphingosine kinase substrates as S1P receptor modulating prodrugs.

In vivo nuclear translocation of mineralocorticoid and glucocorticoid receptors in rat kidney: differential effect of corticosteroids along the distal tubule

Aldosterone and corticosterone bind to mineralocorticoid (MR) and glucocorticoid receptors (GR), which, upon ligand binding, are thought to translocate to the cell nucleus to act as transcription factors. Mineralocorticoid selectivity is achieved by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that inactivates 11β-hydroxy glucocorticoids. High expression levels of 11β-HSD2 characterize the aldosterone-sensitive distal nephron (ASDN), which comprises the segment-specific cells of late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct (CD). We used MR- and GR-specific antibodies to study localization and regulation of MR and GR in kidneys of rats with altered plasma aldosterone and corticosterone levels. In control rats, MR and GR were found in cell nuclei of thick ascending limb (TAL), DCT, CNT, CD cells, and intercalated cells (IC). GR was also abundant in cell nuclei and the subapical compartment of proximal tubule (PT) cells. Dietary NaCl loading, which lowers plasma aldosterone, caused a selective removal of GR from cell nuclei of 11β-HSD2-positive ASDN. The nuclear localization of MR was unaffected. Adrenalectomy (ADX) resulted in removal of MR and GR from the cell nuclei of all epithelial cells. Aldosterone replacement rapidly relocated the receptors in the cell nuclei. In ASDN cells, low-dose corticosterone replacement caused nuclear localization of MR, but not of GR. The GR was redistributed to the nucleus only in PT, TAL, early DCT, and IC that express no or very little 11β-HSD2. In ASDN cells, nuclear GR localization was only achieved when corticosterone was replaced at high doses. Thus ligand-induced nuclear translocation of MR and GR are part of MR and GR regulation in the kidney and show remarkable segment- and cell type-specific characteristics. Differential regulation of MR and GR may alter the level of heterodimerization of the receptors and hence may contribute to the complexity of corticosteroid effects on ASDN function.

A residue close to ?1 loop F disrupts modulation of GABAA receptors by benzodiazepines while their binding is maintained

Benzodiazepines act at the major isoforms of GABA type A receptors where they potentiate the current evoked by the agonist GABA. The underlying mechanism of this potentiation is poorly understood, but hypothesized to be related to the mechanism that links agonist binding to channel opening in these ligand activated ion channels. The loop F of the ?(1) and the ?(2) subunit have been implicated in channel gating, and loop F of the ?(2) subunit in the modulation by benzodiazepines. We have identified the conservative point mutation Y168F located N-terminally of loop F in the ?(1) subunit that fails to affect agonist properties. Interestingly, it disrupts modulation by benzodiazepines, but leaves high affinity binding to the benzodiazepine binding site intact. Modulation by barbiturates and neurosteroids is also unaffected. Residue ?(1) Y168 is not located either near the binding pockets for GABA, or for benzodiazepines, or close to the loop F of the ?(2) subunit. Our results support the fact, that broader regions of ligand gated receptors are conformationally affected by the binding of benzodiazepines. We infer that also broader regions could contribute to signaling from GABA agonist binding to channel opening.

Diversity of structure and function of alpha1alpha6beta3delta GABAA receptors: comparison with alpha1beta3delta and alpha6beta3delta receptors

delta subunit-containing gamma-aminobutyric acid, type A (GABA(A))receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with alpha(1) and/or alpha(6) subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA(A) receptor pentamers by concatenation. These receptors composed of alpha(1), alpha(6), beta(3), and delta subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that delta can assume multiple positions in a receptor pentamer made up of alpha(1), alpha(6), beta(3), and delta subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of delta subunits between two alpha subunits in alpha(1)alpha(6)beta(3)delta receptors. This property is shared by alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors, but there are differences in the additionally expressed isoforms.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease

Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.

Somatostatin receptors as targets for nuclear medicine imaging and radionuclide treatment

Radiolabeled peptides have been an important class of compounds in radiopharmaceutical sciences and nuclear medicine for more than 20 years. Despite strong research efforts, only somatostatin-based radiopeptides have a real impact on patient care, diagnostically and therapeutically. [(111)In-diethylenetriaminepentaacetic acid(0)]octreotide is commercially available for imaging. Imaging was highly improved by the introduction of PET radionuclides such as (68)Ga, (64)Cu, and (18)F. Two peptides are successfully used in targeted radionuclide therapy when bound to DOTA and labeled with (90)Y and (177)Lu.

A closer look at the high affinity benzodiazepine binding site on GABAA receptors

Ligands of the benzodiazepine binding site of the GABA(A) receptor come in three flavors: positive allosteric modulators, negative allosteric modulators and antagonists all of which can bind with high affinity. The GABA(A) receptor is a pentameric protein which forms a chloride selective ion channel and ligands of the benzodiazepine binding site stabilize three different conformations of this protein. Classical benzodiazepines exert a positive allosteric effect by increasing the apparent affinity of channel opening by the agonist γ-aminobutyric acid (GABA). We concentrate here on the major adult isoform, the α(1)β(2)γ(2) GABA(A) receptor. The classical binding pocket for benzodiazepines is located in a subunit cleft between α(1) and γ(2) subunits in a position homologous to the agonist binding site for GABA that is located between β(2) and α(1) subunits. We review here approaches to this picture. In particular, point mutations were performed in combination with subsequent analysis of the expressed mutant proteins using either electrophysiological techniques or radioactive ligand binding assays. The predictive power of these methods is assessed by comparing the results with the predictions that can be made on the basis of the recently published crystal structure of the acetylcholine binding protein that shows homology to the N-terminal, extracellular domain of the GABA(A) receptor. In addition, we review an approach to the question of how the benzodiazepine ligands are positioned in their binding pocket. We also discuss a newly postulated modulatory site for benzodiazepines at the α(1)/β(2) subunit interface, homologous to the classical benzodiazepine binding pocket.

Four-year results following treatment of intrabony periodontal defects with an enamel matrix derivative alone or combined with a biphasic calcium phosphate

The aim of this study was to evaluate the 4-year clinical outcomes following regenerative surgery in intrabony defects with either EMD + BCP or EMD. Twenty-four patients with advanced chronic periodontitis, displaying one-, two-, or three-walled intrabony defect with a probing depth of at least 6 mm, were randomly treated with either EMD + BCP (test) or EMD alone (control). The following clinical parameters were evaluated at baseline, at 1 year and at 4 years after regenerative surgery: plaque index, gingival index, bleeding on probing, probing depth, gingival recession, and clinical attachment level (CAL). The primary outcome variable was CAL. No differences in any of the investigated parameters were observed at baseline between the two groups. The test group demonstrated a mean CAL change from from 10.8 ± 1.6 mm to 7.4 ± 1.6 mm (p < 0.001) and to 7.6 ± 1.7 mm (p < 0.001) at 1 and 4 years, respectively. In the control group, mean CAL changed from 10.4 ± 1.3 at baseline to 6.9 ± 1.0 mm (p < 0.001) at 1 year and 7.2 ± 1.2 mm (p < 0.001) at 4 years. At 4 years, two defects in the test group and three defects in the control group have lost 1 mm of the CAL gained at 1 year. Compared to baseline, at 4 years, a CAL gain of ≥3 mm was measured in 67% of the defects (i.e., in 8 out of 12) in the test group and in 75% of the defects (i.e., in 9 out of 12) in the control group. There were no statistically significant differences in any of the investigated parameters at 1 and at 4 years between the two groups. Within their limits, the present results indicate that: (a) the clinical improvements obtained with both treatments can be maintained over a period of 4 years, and (b) in two- and three-walled intrabony defects, the addition of BCP did not additionally improve the outcomes obtained with EMD alone. In two- and three-walled intrabony defects, the combination of EMD + BCP did not show any advantage over the use of EMD alone.

Oestradiol enhances plasma growth hormone and insulin-like growth factor-I concentrations and increased the expression of their receptors mRNAs in the liver of ovariectomized cows

Many metabolic hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin affect ovarian functions. However, whether ovarian steroid hormones affect metabolic hormones in cattle remains unknown. This study aimed to determine the effect of sex steroids on the plasma profiles of GH, IGF-I and insulin and their receptors in the liver and adipose tissues of dairy cows. Ovariectomized cows (n = 14) were randomly divided into four groups: control group (n = 3) was treated with saline on Day 0; oestradiol (E2) group (n = 3), with saline and 1 mg oestradiol benzoate (EB) on Day 0 and 5, respectively; progesterone (P4) group (n = 4) with two CIDRs (Pfizer Inc., Tokyo, Japan) from Day 0; and E2 + P4 group (n = 4) with two CIDRs on Day 0 that were removed on Day 6 and were immediately injected with 1 mg EB. The animals were euthanized after the experiment, and liver and adipose tissues samples were quantitatively analysed using real-time PCR for the expression of mRNA for the GH (GHR), IGF-I (IGFR-I) and insulin (IR) receptor mRNAs. Oestradiol benzoate significantly increased the number of peaks (p < 0.05), pulse amplitude (p < 0.05) and area under the curve (AUC; p < 0.01) for plasma GH; moreover, it increased plasma IGF-I concentration (p < 0.05), but it had no effect on the plasma insulin profile. P4 significantly decreased the AUC (p < 0.01), compared with the control group, whereas it did not affect the number of peaks and the amplitude of GH pulses. P4 + E2 did not affect the GH pulse profile. E2 increased the mRNA expression of GHR, IGFR-I and IR in the liver (p < 0.05), whereas both P4 and E2 + P4 did not change their expressions. Our results provide evidence that the metabolic and reproductive endocrine axes may regulate each other to ensure optimal reproductive and metabolic function.

First continuous phosphate record from Greenland ice cores

A continuous and highly sensitive absorption method for detection of soluble phosphate in ice cores has been developed using a molybdate reagent and a 2m liquid waveg- uide (LWCC). The method is optimized to meet the low concentrations of phosphate in Greenland ice, it has a detection limit of around 0.1ppb and a depth resolution of approximately 2cm. The new method has been applied to obtain phosphate concen- trations from segments of two Northern Greenland ice cores: from a shallow firn core covering the most recent 120yr and from the recently obtained deep NEEM ice core in which sections from the late glacial period have been analysed. Phosphate con- centrations in 20th century ice are around 0.32ppb with no indication of anthropogenic influence in the most recent ice. In the glacial part of the NEEM ice core concentra- tions in the cold stadial periods are significantly higher, in the range of 6–24ppb, while interstadial ice concentrations are around 2ppb. In the shallow firn core, a strong cor- relation between concentrations of phosphate and insoluble dust suggests a similar deposition pattern for phosphate and dust. In the glacial ice, phosphate and dust also correlate quite strongly, however it is most likely that this correlation originates from the phosphate binding to dust during transport, with only a fraction coming directly from dust. Additionally a constant ratio between phosphate and potassium concentrations shows evidence of a possible biogenic land source.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Altering the sphingosine-1-phosphate/ceramide balance: a promising approach for tumor therapy

In recent years sphingolipids have emerged as important signaling molecules regulating fundamental cell responses such as cell death and differentiation, proliferation and aspects of inflammation. Especially ceramide has been a main focus of research since it possesses pro-apoptotic capacity in many cell types. A counterplayer of ceramide was found in sphingosine-1-phosphate (S1P), which is generated from ceramide by the consecutive actions of ceramidase and sphingosine kinase. S1P can potently induce cell proliferation via binding to and activation of the Edg family of receptors which have now been renamed as S1P receptors. Obviously, a delicate balance between ceramide and sphingosine-1-phosphate determines whether cells undergo apoptosis or proliferate, two cell responses that are critically involved in tumor development. Directing the balance in favor of ceramide, i.e. by inhibiting ceramidase or sphingosine kinase activities may support the pro-apoptotic action of ceramide and thus may have beneficial effects in cancer therapy. This review will summarize novel insights into the regulation of sphingolipid formation and their potential involvement in tumor development. Finally, we will pinpoint potential new targets for tumor therapy.