Effects of enamel matrix proteins in combination with a bovine-derived natural bone mineral for the repair of bone defects.

OBJECTIVES Previously, the use of enamel matrix derivative (EMD) in combination with a natural bone mineral (NBM) was able to stimulate periodontal ligament cell and osteoblast proliferation and differentiation. Despite widespread use of EMD for periodontal applications, the effects of EMD on bone regeneration are not well understood. The aim of the present study was to test the ability of EMD on bone regeneration in a rat femur defect model in combination with NBM. MATERIALS AND METHODS Twenty-seven rats were treated with either NBM or NBM + EMD and assigned to histological analysis at 2, 4, and 8 weeks. Defect morphology and mineralized bone were assessed by μCT. For descriptive histology, hematoxylin and eosin staining and Safranin O staining were performed. RESULTS Significantly more newly formed trabecular bone was observed at 4 weeks around the NBM particles precoated with EMD when compared with NBM particles alone. The drilled control group, in contrast, achieved minimal bone regeneration at all three time points (P < 0.05). CONCLUSIONS The present results may suggest that EMD has the ability to enhance the speed of new bone formation when combined with NBM particles in rat osseous defects. CLINICAL RELEVANCE These findings may provide additional clinical support for the combination of EMD with bone graft for the repair of osseous and periodontal intrabony defects.

Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms--which stain is suitable?

BACKGROUND There is confusion over the definition of the term "viability state(s)" of microorganisms. "Viability staining" or "vital staining techniques" are used to distinguish live from dead bacteria. These stainings, first established on planctonic bacteria, may have serious shortcomings when applied to multispecies biofilms. Results of staining techniques should be compared with appropriate microbiological data. DISCUSSION Many terms describe "vitality states" of microorganisms, however, several of them are misleading. Authors define "viable" as "capable to grow". Accordingly, staining methods are substitutes, since no staining can prove viability.The reliability of a commercial "viability" staining assay (Molecular Probes) is discussed based on the corresponding product information sheet: (I) Staining principle; (II) Concentrations of bacteria; (III) Calculation of live/dead proportions in vitro. Results of the "viability" kit are dependent on the stains' concentration and on their relation to the number of bacteria in the test. Generally this staining system is not suitable for multispecies biofilms, thus incorrect statements have been published by users of this technique.To compare the results of the staining with bacterial parameters appropriate techniques should be selected. The assessment of Colony Forming Units is insufficient, rather the calculation of Plating Efficiency is necessary. Vital fluorescence staining with Fluorescein Diacetate and Ethidium Bromide seems to be the best proven and suitable method in biofilm research.Regarding the mutagenicity of staining components users should be aware that not only Ethidium Bromide might be harmful, but also a variety of other substances of which the toxicity and mutagenicity is not reported. SUMMARY - The nomenclature regarding "viability" and "vitality" should be used carefully.- The manual of the commercial "viability" kit itself points out that the kit is not suitable for natural multispecies biofilm research, as supported by an array of literature.- Results obtained with various stains are influenced by the relationship between bacterial counts and the amount of stain used in the test. Corresponding vitality data are prone to artificial shifting.- As microbiological parameter the Plating Efficiency should be used for comparison.- Ethidium Bromide is mutagenic. Researchers should be aware that alternative staining compounds may also be or even are mutagenic.

Natural history of surgically treated high-risk prostate cancer

BACKGROUND No data exist on the patterns of biochemical recurrence (BCR) and their effect on survival in patients with high-risk prostate cancer (PCa) treated with surgery. The aim of our investigation was to evaluate the natural history of PCa in patients treated with radical prostatectomy (RP) alone. MATERIALS AND METHODS Overall, 2,065 patients with high-risk PCa treated with RP at 7 tertiary referral centers between 1991 and 2011 were identified. First, we calculated the probability of experiencing BCR after surgery. Particularly, we relied on conditional survival estimates for BCR after RP. Competing-risks regression analyses were then used to evaluate the effect of time to BCR on the risk of cancer-specific mortality (CSM). RESULTS Median follow-up was 70 months. Overall, the 5-year BCR-free survival rate was 55.2%. Given the BCR-free survivorship at 1, 2, 3, 4, and 5 years, the BCR-free survival rates improved by+7.6%,+4.1%,+4.8%,+3.2%, and+3.7%, respectively. Overall, the 10-year CSM rate was 14.8%. When patients were stratified according to time to BCR, patients experiencing BCR within 36 months from surgery had higher 10-year CSM rates compared with those experiencing late BCR (19.1% vs. 4.4%; P<0.001). At multivariate analyses, time to BCR represented an independent predictor of CSM (P<0.001). CONCLUSIONS Increasing time from surgery is associated with a reduction of the risk of subsequent BCR. Additionally, time to BCR represents a predictor of CSM in these patients. These results might help provide clinicians with better follow-up strategies and more aggressive treatments for early BCR.

Natural course of pontocerebellar hypoplasia type 2A

INTRODUCTION Pontocerebellar hypoplasia Type 2 (PCH2) is a rare autosomal recessive condition, defined on MRI by a small cerebellum and ventral pons. Clinical features are severe developmental delay, microcephaly and dyskinesia.Ninety percent carry a p.A307S mutation in the TSEN54-gene. Our aim was to describe the natural course including neurological and developmental features and other aspects of care in a homogeneous group of PCH2 patients all carrying the p.A307S mutation. PATIENTS AND METHODS Patients were recruited via the German patients' organizations. Inclusion criteria were imaging findings of PCH2 and a p.A307S mutation. Data were collected using medical reports and patient questionnaires discussed in a standardized telephone interview. RESULTS Thirty-three patients were included. When considering survival until age 11 years, 53% of children had died Weight, length and head circumference, mostly in the normal range at birth, became abnormal, especially head circumference (-5.58 SD at age 5 yrs). Neurologic symptoms: Choreathetosis was present in 88% (62% with pyramidal signs), 12% had pure spasticity. Epileptic seizures were manifest in 82%, status epilepticus in 39%. Non-epileptic dystonic attacks occurred in 33%. General symptoms: feeding difficulties were recorded in 100%, sleep disorder in 96%, apneas in 67% and recurrent infections in 52%; gastroesophageal reflux disease was diagnosed in 73%, 67% got percutaneous endoscopic gastrostomy and 36% a Nissen-fundoplication. Neurodevelopmental data: All children made progress, but on a low level: such as fixing and following with the eyes was seen in 76%, attempting to grasp objects (76%), moderate head control (73%), social smile (70%), rolling from prone to supine (58%), and sitting without support (9%). Ten percent lost achieved abilities on follow-up. The presence of prenatal symptoms did not correlate with outcome. CONCLUSION Phenotype of this genetically homogeneous group of PCH2 children was severe with reduced survival, but compatible with some developmental progress. Our data support the hypothesis of an early onset degeneration which thereafter stabilizes.

Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABA(A) receptors.

Biphenylic compounds related to the natural products magnolol and 4'-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA(A) receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5'-hexylbiphenyl-2,2'-diol (45) and the honokiol analogs 4'-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4'-methoxybiphenyl-2-ol (62) and 5-hexyl-4'-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA(A) receptor agonists.

Man-induced activities modify demographic parameters in a long-lived species: effects of poisoning and health policies

Recent changes in sanitary policies within the European Union (EU) concerning disposal of carcasses of domestic animals and the increase of non-natural mortality factors, such as illegal poisoning, are threatening European vultures. However, the effects of anthropogenic activities on demographic parameters are poorly studied. Using a long-term study (1994–2011) of the threatened Pyrenean Bearded Vulture Gypaetus barbatus population, we assess the variation in the proportion of breeding pairs, egg-laying dates, clutch size, breeding success, and survival following a sharp reduction in food availability in 2005 due to the application of restrictive sanitary policies decreasing livestock carcass availability. We found a delay in laying dates and a regressive trend in clutch size, breeding success, and survival following policy change. The maintenance of specific supplementary feeding stations for Bearded Vultures probably reduced the negative effects of illegal poisoning and food shortages, which mainly affected subadult survival. A drop in food availability may have produced changes in demographic parameters and an increase in mortality due to an increased exposure to contaminated food. As a result, supplementary feeding as a precautionary measure can be a useful tool to reduce illegal poisoning and declines in demographic parameters until previous food availability scenarios are achieved. This study shows how anthropogenic activities through human health regulations that affect habitat quality can suddenly modify demographic parameters in long-lived species, including those, such as survival, with high sensitivity to population growth rate.

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

One-pot heterogeneous synthesis of Δ(3)-tetrahydrocannabinol analogues and xanthenes showing differential binding to CB(1) and CB(2) receptors

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive cannabinoid in hemp (Cannabis sativa L.) and responsible for many of the pharmacological effects mediated via cannabinoid receptors. Despite being the major cannabinoid scaffold in nature, Δ(9)-THC double bond isomers remain poorly studied. The chemical scaffold of tetrahydrocannabinol can be assembled from the condensation of distinctly substituted phenols and monoterpenes. Here we explored a microwave-assisted one pot heterogeneous synthesis of Δ(3)-THC from orcinol (1a) and pulegone (2). Four Δ(3)-THC analogues and corresponding Δ(4a)-tetrahydroxanthenes (Δ(4a)-THXs) were synthesized regioselectively and showed differential binding affinities for CB1 and CB2 cannabinoid receptors. Here we report for the first time the CB1 receptor binding of Δ(3)-THC, revealing a more potent receptor binding affinity for the (S)-(-) isomer (hCB1Ki = 5 nM) compared to the (R)-(+) isomer (hCB1Ki = 29 nM). Like Δ(9)-THC, also Δ(3)-THC analogues are partial agonists at CB receptors as indicated by [(35)S]GTPγS binding assays. Interestingly, the THC structural isomers Δ(4a)-THXs showed selective binding and partial agonism at CB2 receptors, revealing a simple non-natural natural product-derived scaffold for novel CB2 ligands.

Modified natural cycle in vitro fertilization an alternative in vitro fertilization treatment with lower costs per achieved pregnancy but longer treatment time

OBJECTIVE To analyze the cost and time requirement per achieved pregnancy in optimized modified natural cycle in vitro fertilization (mNC-IVF) based on a treatment protocol with very few consultations and to compare those with conventional gonadotropin-stimulated aVF (clVF) cycles. STUDY DESIGN Mono centric prospective trial. Eighty infertile patients each received 1 modified mNC-IVF cycle using low doses of the clomiphene citrate. Based on the number of consultations and the clinical pregnancy rate per cycle, the total costs and required time to achieve a pregnancy were analyzed and compared with cIVF. Calculations for cIVF were based on standard therapy protocols and outcomes of European registries. RESULTS Patients (21-42 years old, 35.4 +/- 4.7 years) undergoing mNC-IVF required on average 1.2 consultations before follicle aspiration. Pregnancy rate per transfer and per initiated cycle were 25% and 13.6%, respectively. Multiple pregnancies did not occur. According to the calculations, total costs per pregnancy rate were around 15% lower with mNC-IVF as compared to cIVF. In contrast, time to achieve an equal pregnancy rate was calculated to take around 30% longer with mNC-IVF as compared to cIVF. CONCLUSION mNC-IVF using very low dosages of clomiphene citrate avoids multiple pregnancies and is less expensive but more time consuming per achieved pregnancy when compared to clVF.

Serum but not follicular fluid cytokine levels are increased in stimulated versus natural cycle IVF: a multiplexed assay study.

Throughout follicular growth the number of immune cells increases, enhanced under stimulation with exogenous gonadotropins. This treatment, however, may adversely influence folliculogenesis and negatively affect oocyte quality through modifications in the follicular concentrations of cytokines released by these immune cells. We studied this hypothesis by systematically analysing the concentrations of cytokines present in the serum and follicular fluid at the time of follicular aspiration in conventional gonadotropin-stimulated (c-IVF) cycles in comparison with natural cycle IVF (NC-IVF) in which the follicles were naturally matured. Our study involved 37 NC-IVF and 39 c-IVF cycles including 13 women who underwent both therapies. Mean age was 35.3 ± 4.6 (SD) and 34.2 ± 3.7 years in the NC-IVF and c-IVF groups (ns). Thirteen cytokines were determined in matched serum and FF samples. Interleukin (IL)-4, TNF-α, RANTES, eotaxin and interferon-gamma-induced protein-10 concentrations were lower in FF than in serum. IL-6, -8, -10, -18, monocyte chemotactic protein-1 (MCP-1), VEGF and leukaemia inhibitory factor (LIF) showed higher median levels in FF than in serum, indicating possible ovarian production. Most of these markers were also increased in concentration in the stimulated (c-IVF) than in the NC groups in the serum, but not in the follicular fluid. This finding can be attributed to the increased number of active follicles present after controlled ovarian stimulation. IL-8 was reduced in c-IVF cycles. Our study did not reveal differences in follicular fluid but in serum cytokine concentrations, suggesting that the follicular immune system might not be significantly affected by gonadotropin stimulation.

Low-dosage clomiphene reduces premature ovulation rates and increases transfer rates in natural-cycle IVF

Natural-cycle IVF has been suggested as an alternative IVF treatment. However, efficacy is limited due to high premature ovulation rates, resulting in low transfer rates. This study investigates whether low dosages of clomiphene citrate reduce premature ovulation rate and increase transfer rate. Of 112 women included (aged 35.2 ± 4.5 years) 108 underwent one natural-cycle IVF cycle with human chorionic gonadotrophin (HCG) to induce ovulation and 103 underwent one natural-cycle IVF cycle with 25 mg/day clomiphene from about day 7 until HCG administration. Before retrieval, 1.2 monitoring consultations per cycle were required. Clomiphene reduced premature ovulation rate, from 27.8% without to 6.8% with clomiphene (P < 0.001) and increased transfer rate from 39.8% to 54.4% (P = 0.039). Clinical pregnancy rates without and with clomiphene were 27.9% versus 25.0% per transfer and 11.1% versus 13.6% per initiated cycle. Use of clomiphene resulted in mild hot flushes and headache in 5% of patients. Nausea and persisting ovarian cyst formation was not observed. In conclusion, clomiphene citrate led to very few side effects, required 1.2 monitoring consultations, significantly reduced premature ovulation rate and significantly increased transfer rate per initiated cycle, an effect which was not age dependent.

Natural anti-FcepsilonRIalpha autoantibodies may interfere with diagnostic tests for autoimmune urticaria.

IgG autoantibodies against the alpha-chain of the high affinity IgE receptor are claimed to play a pathogenetic role in autoimmune urticaria. The best methods for detection of functional autoantibodies are currently the autologous serum skin test and the basophil histamine release assay. A simplified and feasible screening test would facilitate the diagnosis of autoimmune urticaria. Here we offer an explanation for the difficulties in establishing a screening test for autoantibodies directed against the alpha-chain of the high affinity IgE receptor in autoimmune urticaria. Identical autoantibodies in chronic urticaria patients and healthy donors belonging to the natural autoantibody repertoire were found by sequence analysis of anti-alpha-chain autoantibodies isolated by repertoire cloning from antibody libraries. These natural autoantibodies bound to the receptor and triggered histamine release but only if IgE was previously removed from the receptor. Diagnostic assays used for detection of antibodies directed against the IgE receptor may require signal comparison with and without the artificial removal of IgE, immune complexes, and complement in order to avoid false positive or negative results. After IgE removal diagnostic tests will detect natural autoantibodies against the high affinity IgE receptor regardless of whether they are pathogenic or not.

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.