Direct experimental evidence that early-life farm environment influences regulation of immune responses

Background: In mammals, early-life environmental variations appear to affect microbial colonization and therefore competent immune development, and exposure to farm environments in infants has been inversely correlated with allergy development. Modelling these effects using manipulation of neonatal rodents is difficult due to their dependency on the mother, but the relatively independent piglet is increasingly identified as a valuable translational model for humans. This study was designed to correlate immune regulation in piglets with early-life environment. Methods: Piglets were nursed by their mother on a commercial farm, while isolatorreared siblings were formula fed. Fluorescence immunohistology was used to quantify T-reg and effector T-cell populations in the intestinal lamina propria and the systemic response to food proteins was quantified by capture ELISA. Results: There was more CD4+ and CD4+CD25+ effector T-cell staining in the intestinal mucosa of the isolator-reared piglets compared with their farm-reared counterparts. In contrast, these isolator-reared piglets had a significantly reduced CD4+CD25+Foxp3+ regulatory T-cell population compared to farm-reared littermates, resulting in a significantly higher T-reg-to-effector ratio in the farm animals. Consistent with these findings, isolator-reared piglets had an increased serum IgG anti-soya response to novel dietary soya protein relative to farm-reared piglets. Conclusion: Here, we provide the first direct evidence, derived from intervention, that components of the early-life environment present on farms profoundly affects both local development of regulatory components of the mucosal immune system and immune responses to food proteins at weaning. We propose that neonatal piglets provide a tractable model which allows maternal and treatment effects to be statistically separated.

A comparison of methods for constructing confidence intervals after phase II/III clinical trials

Recently, in order to accelerate drug development, trials that use adaptive seamless designs such as phase II/III clinical trials have been proposed. Phase II/III clinical trials combine traditional phases II and III into a single trial that is conducted in two stages. Using stage 1 data, an interim analysis is performed to answer phase II objectives and after collection of stage 2 data, a final confirmatory analysis is performed to answer phase III objectives. In this paper we consider phase II/III clinical trials in which, at stage 1, several experimental treatments are compared to a control and the apparently most effective experimental treatment is selected to continue to stage 2. Although these trials are attractive because the confirmatory analysis includes phase II data from stage 1, the inference methods used for trials that compare a single experimental treatment to a control and do not have an interim analysis are no longer appropriate. Several methods for analysing phase II/III clinical trials have been developed. These methods are recent and so there is little literature on extensive comparisons of their characteristics. In this paper we review and compare the various methods available for constructing confidence intervals after phase II/III clinical trials.

Methods for the modulation and analysis of NF-κB-dependent adult neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-κB plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-κB activity can be used. In this study, methods are described for the analysis of NF-κB-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies

Intracellular reactive oxygen species (ROS) production is essential to normal cell function. However, excessive ROS production causes oxidative damage and cell death. Many pharmacological compounds exert their effects on cell cycle progression by changing intracellular redox state and in many cases cause oxidative damage leading to drug cytotoxicity. Appropriate measurement of intracellular ROS levels during cell cycle progression is therefore crucial in understanding redox-regulation of cell function and drug toxicity and for the development of new drugs. However, due to the extremely short half-life of ROS, measuring the changes in intracellular ROS levels during a particular phase of cell cycle for drug intervention can be challenging. In this article, we have provided updated information on the rationale, the applications, the advantages and limitations of common methods for screening drug effects on intracellular ROS production linked to cell cycle study. Our aim is to facilitate biomedical scientists and researchers in the pharmaceutical industry in choosing or developing specific experimental regimens to suit their research needs.

Qualitative tools & experimental philosophy

Experimental philosophy brings empirical methods to philosophy. These methods are used to probe how people think about philosophically interesting things such as knowledge, morality, freedom, etc. This paper explores the contribution that qualitative methods have to make in this enterprise. I argue that qualitative methods have the potential to make a much greater contribution than they have so far. Along the way, I acknowledge a few types of resistance that proponents of qualitative methods in experimental philosophy might encounter, and provide reasons to think they are ill-founded.

Mesenchymal Stem Cells and Bovine Bone Mineral in Sinus Lift Procedures-An Experimental Study in Sheep

Introduction: New reconstructive and less invasive methods have been searched to optimize bone formation and osseointegration of dental implants in maxillary sinus augmentation. Purpose: The aim of the presented ovine split-mouth study was to compare bovine bone mineral (BBM) alone and in combination with mesenchymal stem cells (MSCs) regarding their potential in sinus augmentation. Material and Methods: Bilateral sinus floor augmentations were performed in six adult sheep. BBM and MSCs were placed into the test side and only BBM in the contra-lateral control side of each sheep. Animals were sacrificed after 8 and 16 weeks. Augmentation sites were analyzed by computed tomography, histology, and histomorphometry. Results: The initial volumes of both sides were similar and did not change significantly with time. A tight connection between the particles of BBM and the new bone was observed histologically. Bone formation was significantly (p = 0.027) faster by 49% in the test sides. Conclusion: The combination of BBM and MSCs accelerated new bone formation in this model of maxillary sinus augmentation. This could allow early placement of implants.

Hard tissue formation adjacent to implants of various size and configuration immediately placed into extraction sockets: an experimental study in dogs

Objectives To evaluate the influence of implant size and configuration on osseointegration in implants immediately placed into extraction sockets. Material and methods Implants were installed immediately into extraction sockets in the mandibles of six Labrador dogs. In the control sites, cylindrical transmucosal implants (3.3 mm diameter) were installed, while in the test sites, larger and conical (root formed, 5 mm diameter) implants were installed. After 4 months of healing, the resorptive patterns of the alveolar crest were evaluated histomorphometrically. Results With one exception, all implants were integrated in mineralized bone, mainly composed of mature lamellar bone. The alveolar crest underwent resorption at the control as well as at the test implants. This resorption was more pronounced at the buccal aspects and significantly greater at the test (2.7 +/- 0.4 mm) than at the control implants (1.5 +/- 0.6 mm). However, the control implants were associated with residual defects that were deeper at the lingual than at the buccal aspects, while these defects were virtually absent at test implants. Conclusions The installment of root formed wide implants immediately into extraction sockets will not prevent the resorption of the alveolar crest. In contrast, this resorption is more marked both at the buccal and lingual aspects of root formed wide than at standard cylindrical implants. To cite this article:Caneva M, Salata LA, de Souza SS, Bressan E, Botticelli D, Lang NP. Hard tissue formation adjacent to implants of various size and configuration immediately placed into extraction sockets: an experimental study in dogs.Clin. Oral Impl. Res. 21, 2010; 885-895.doi: 10.1111/j.1600-0501.2010.01931.x.

Influence of lateral pressure to the implant bed on osseointegration: an experimental study in dogs

Aim To study osseointegration and bone-level changes at implants installed using either a standard or a reduced diameter bur for implant bed preparation. Material and methods In six Labrador dogs, the first and second premolars were extracted bilaterally. Subsequently, mesial roots of the first molars were endodontically treated and distal roots, including the corresponding part of the crown, were extracted. After 3 months of healing, flaps were elevated and recipient sites were prepared in all experimental sites. The control site was prepared using a standard procedure, while the test site was prepared using a drill with a 0.2 mm reduced diameter than the standard one used in the contra-lateral side. After 4 months of healing, the animals were euthanized and biopsies were obtained for histological processing and evaluation. Results With the exception of one implant that was lost, all implants were integrated in mineralized bone. The alveolar crest underwent resorption at control as well as at test sites (buccal aspect similar to 1 mm). The most coronal contact of bone-to-implant was located between 1.2 and 1.6 mm at the test and between 1.3 and 1.7 mm at the control sites. Bone-to-implant contact percentage was between 49% and 67%. No statistically significant differences were found for any of the outcome variables. Conclusions After 4 months of healing, lateral pressure to the implant bed as reflected by higher insertion torques (36 vs. 15 N cm in the premolar and 19 vs. 7 N cm in the molar regions) did not affect the bone-to-implant contact. To cite this article:Pantani F, Botticelli D, Garcia IR Jr., Salata LA, Borges GJ, Lang NP. Influence of lateral pressure to the implant bed on osseointegration: an experimental study in dogs.Clin. Oral Impl. Res. 21, 2010; 1264-1270.doi: 10.1111/j.1600-0501.2009.01941.x.

Effects of Early Functional Loading on Maintenance of Free Autogenous Bone Graft and Implant Osseointegration: An Experimental Study in Dogs

Purpose: The aim of the present study was to investigate the healing, integration, and maintenance of autogenous onlay bone grafts and implant osseointegration either loaded in the early or the delayed stages. Materials and Methods: A total of 5 male clogs received bilateral blocks of onlay bone grafts harvested from the contralateral alveolar ridge of the mandible. On one side, the bone block was secured by 3 dental implants (3.5 mm x 13.0 mm, Osseospeed; Astra Tech AB, Molndal, Sweden). Two implants at the extremities of the graft were loaded 2 clays after installation by abutment connection and prosthesis (simultaneous implant placement group); the implant in the middle remained unloaded and served as the control. On the other side, the block was fixed with 2 fixation screws inserted in the extremities of the graft. Four weeks later, the fixation screws were replaced with 3 dental implants. The loading procedure (delayed implant placement group) was performed 2 clays later, as described for the simultaneous implant placement sites. The animals were sacrificed 12 weeks after the grafting procedure. Implant stability was measured through resonance frequency analysis. The bone volume and density were assessed on computed tomography. The bone to implant contact and bone area in a region of interest were evaluated on histologic slides. Results: The implant stability quotient showed statistical significance in favor of the delayed loaded grafts (P=.001). The bone-to-implant contact (P=.008) and bone area in a region of interest (P=0.005) were significantly greater in the delayed group. Nevertheless, no difference was found in terms of graft volume and density between the early loaded and delayed-loaded approaches. Conclusions: The protocol in which the implant and bone graft were given delayed loading allows for effective quality of implant osseointegration and stabilization, with healing and remodeling occurring in areas near the implant resulting in denser bone architecture. (C) 2010 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Sing 68:825-832, 2010

EFFECT OF DRYING METHODS ON THE THERMODYNAMIC PROPERTIES OF BLACKBERRY PULP POWDER

Three different types of maltodextrin encapsulated dehydrated blackberry fruit powders were obtained using vibrofluidized bed drying (VF), spray drying (SD), vacuum drying (VD), and freeze drying (FD). Moisture equilibrium data of blackberry pulp powders with 18% maltodextrin were determined at 20, 30, 40, and 50 degrees C using the static gravimetric method for the water activity range of 0.06-0.90. Experimental equilibrium moisture content data versus water activity were fit to the Guggenheim-Anderson-de Boer (GAB) model. Agreement was found between experimental and calculated values. The isosteric heat of sorption of water was determined using the Clausius-Clapeyron equation from the equilibrium data; isosteric heats of sorption were found to increase with increasing temperature and could be adjusted by an exponential relationship. For freeze dried, vibrofluidized, and vacuum dried pulp powder samples, the isosteric heats of sorption were lower (more negative) than those calculated for spray dried samples. The enthalpy-entropy compensation theory was applied to sorption isotherms and plots of Delta H versus Delta S provided the isokinetic temperatures, indicating an enthalpy-controlled sorption process.

Effects of exercise training on autonomic dysfunction management in an experimental model of menopause and myocardial infarction

Objective: The aim of this study was to investigate the effects of exercise training on cardiovascular autonomic dysfunction in ovariectomized rats submitted to myocardial infarction. Methods: Female Wistar rats were divided into the following ovariectomized groups: sedentary ovariectomized (SO), trained ovariectomized (TO), sedentary ovariectomized infarcted (SOI), and trained ovariectomized infarcted (TOI). Trained groups were submitted to an exercise training protocol on a treadmill (8 wk). Arterial baroreflex sensitivity was evaluated by heart rate responses to arterial pressure changes, and cardiopulmonary baroreflex sensitivity was tested by bradycardic and hypotension responses to serotonin injection. Vagal and sympathetic effects were calculated by pharmacological blockade. Results: Arterial pressure was reduced in the TO in comparison with the SO group and increased in the TOI in relation to the SOI group. Exercise training improved the baroreflex sensitivity in both the TO and TOI groups. The TOI group displayed improvement in cardiopulmonary reflex sensitivity compared with the SOI group at the 16 mu g/kg serotonin dose. Exercise training enhanced the vagal effect in both the TO (45%) and TOI (46%) animals compared with the SO and SOI animals and reduced the sympathetic effect in the TOI (38%) in comparison with the SOI animals. Significant correlations were obtained between bradycardic baroreflex responses and vagal (r = -0.7, P < 0.005) and sympathetic (r = 0.7, P < 0.001) effects. Conclusions: These results indicate that exercise training in ovariectomized rats submitted to myocardial infarction improves resting hemodynamic status and reflex control of the circulation, which may be due to an increase in the vagal component. This suggests a homeostatic role for exercise training in reducing the autonomic impairment of myocardial infarction in postmenopausal women.

A light and scanning electron microscopy study of bone healing following inferior alveolar nerve lateralization: An experimental study in rabbits

Purpose: The purpose of this study was to evaluate the bone healing kinetics around commercially pure titanium implants following inferior alveolar nerve (IAN) lateralization in a rabbit model. Materials and Methods: Inferior alveolar nerve lateralization was performed in 16 adult female rabbits (Oryctolagus cuniculus). During the nerve lateralization procedure, 1 implant was placed through the mandibular canal, and the IAN was replaced in direct contact with the implant. During the 8-week healing period, various bone labels were administered for fluorescent microscopy analysis. The animals were euthanized by anesthesia overdose, and the mandibular blocks were exposed by sharp dissection. Nondecalcified samples were prepared for optical light and scanning electron microscopy (SEM) evaluation. Results: SEM evaluation showed bone modeling/remodeling between the IAN and implant surface. Fluorochrome area fraction labeling at different times during the healing period showed that bone apposition mainly occurred during the first 2 weeks after implantation. Conclusions: The results obtained showed that bone healing/deposition occurred between the alveolar nerves in contact with a commercially pure titanium implant. No interaction between the nerve and the implant was detected after the 8-week healing period. Appositional bone healing occurred around the nerve bundle structure, restoring the mandibular canal integrity and morphology.

HISTOLOGIC EVALUATION OF END-TO-SIDE NEURORRHAPHY. EXPERIMENTAL STUDY IN RATS

Objective: The aim of this study was to histologically compare the axonal sprouting after end-to-side neurorrhaphy with or without epineurotomy. Methods: twenty male Wistar rats were used, divided into two groups of 10 rats each. A 1.0cm segment of the tibial nerve E was dried and sutured on the opposite side, where it was sutured into the sciatic nerve D. In Group I, the suture was made directly in the epineurium and in Group II, epineurotomy was performed. After 4 weeks, histological evaluation was carried out of the transposed segment and the sciatic nerve distal to the suture. Results: the results showed a small number of remyelinated fibers, varying from 7 to 51 fibers in Group I and from 10 to 91 fibers in Group II. The Mann-Whitney U test was used, with p=0.311, showing there is no statistically significant difference between the two groups. There was no positive relation between the number of remyelinated fibers in the graft and in the suture site distal to the sciatic lesion. Conclusion: lateral-ending neurorrhaphy, with or without epineural window, does not promote efficient remyelinization. Level of Evidence: Level II, prospective comparative study.

p38 alpha MAP Kinase Controls IL-17 Synthesis in Vogt-Koyanagi-Harada Syndrome and Experimental Autoimmune Uveitis

PURPOSE. Interleukin (IL)-17, which is responsible for the initial influx of leukocytes into the target tissue, was recently described as the main cytokine involved in autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is a significant cause of noninfectious blindness in the world. Herein the authors aimed at unraveling the involvement of IL-17 in VKH and in experimental autoimmune uveitis, focusing on the signaling pathways involved in IL-17 synthesis. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin. Draining lymph node cells, harvested 21 days after immunization, were cultured in the presence or absence of p38 alpha mitogen-activated protein kinase (MAPK) inhibitor (SB203580) and assayed for cytokine production and quantification of CD4(+)IL-17(+) cells. Mice received intraocular injections of SB203580, and disease severity was evaluated by histologic examination of the enucleated eyes at day 21. CD4(+) lymphocytes from MSK-1/2-deficient mice, human CD4(+) cells silenced with MSK1 siRNA, or peripheral blood mononuclear cells (PBMCs) from VKH patients were cultured in the presence or absence of p38 alpha MAPK inhibitor and then assayed for IL-17, IFN-gamma, and IL-4 production. RESULTS. The inhibition of p38 alpha MAPK fully blocked the synthesis of IL-17 by PBMCs from VKH patients and lymphocytes from EAU mice. The absence of the msk1/2 gene resulted in failure to produce IL-17 by murine and human lymphocytes. Interestingly, intraocular injections of SB203580 in EAU mice did not suppress development of the disease. CONCLUSIONS. These data show that p38 alpha MAPK-MSK1/2 is involved in the control of IL-17 synthesis by CD4(+) T cells and that inhibition of p38 alpha MAPK in vitro suppresses IL-17 synthesis but that inhibition of this kinase in vivo did not protect from EAU. (Invest Ophthalmol Vis Sci. 2010;51:3567-3574) DOI: 10.1167/iovs.09-4393

Evaluation of Experimental Autoimmune Uveitis in Mice Treated with FTY720

PURPOSE. FTY720 (fingolimod) is an immunomodulatory drug capable of preventing T-cell migration to inflammatory sites by binding to and subsequently downregulating the expression of sphingosine-1 phosphate receptor 1 (S1P(1)) leading in turn to T-cell retention in lymphoid organs. Additional effects of FTY720 by increasing functional activity of regulatory T cells have recently been demonstrated, raising the conversion of conventional T cells into regulatory T cells and affecting the sequestration of regulatory T cells in normal mice. In this study, the action of FTY720 in the ocular autoimmune model in mice was investigated. METHODS. Mice were immunized with 161-180 peptide and pertussis toxin and were treated with 1 mg/kg/d FTY720 by gavage (7-21 days postimmunization [dpi]) or left untreated. Spleen cells, harvested 21 dpi, were cultured and assayed for cytokine production. Draining lymph node, spleen, and eye cells 21 dpi were assayed for quantification of T-cell populations. Disease severity was evaluated by histologic examination of the enucleated eyes at 21 and 49 dpi. In addition, anti-IRBP antibodies were analyzed by ELISA. RESULTS. FTY720 was effective in suppressing the experimental autoimmune uveitis score. Although there was a reduction in the number of eye-infiltrating cells, FTY did not prevent Treg accumulation at this site. FTY720 leads to a significant increase of CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cell percentages in lymph nodes, suggesting that this site could be the source of Treg cells found in the eye. CONCLUSIONS. The data showed that treatment in vivo with FTY720 was able to suppress EAU in mice. These results are indicative of the possible therapeutic use of FTY720 in ocular autoimmune processes. (Invest Ophthalmol Vis Sci. 2010;51:2568-2574) DOI:10.1167/iovs.09-4769