Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated.2, 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery.Hind-limb ischemia was induced in 10–12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mmHigher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent.Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium. Peripheral and coronary vascular disease still represent a major socio-economic health burden in industrialized countries. The effect of surgical or interventional revascularization is limited due to nonviable myocytes. Therefore clinicians are still in need of regenerative therapies as an adjunct to their treatment armentarium. Current treatment strategies for regeneration of infarcted muscle include (stem) cell or gene therapy based approaches. However, none of them yet gained broad clinical use due to distinct limitations A clinically safe and feasible regenerative therapy could dramatically improve outcome of patients as well as their long-term quality of life. Shock wave treatment is used in medicine for over 30 years for lithotripsy Furthermore, low energy shock wave treatment (SWT) has been shown to cause relief of angina symptoms in patients suffering from coronary artery disease de novo vessel formation (vasculogenesis) via the SDF-1/CXCR4 axis is involved in neovascularization after shock wave treatment of ischemic muscle In a recent work we have been able to proof that not only sprouting of existing vessels (angiogenesis), but as well the recruitment of endothelial progenitor cells for Shock wave therapy is developing for a large variety of indications, including acute and chronic soft tissue wounds Having been shown to induce angiogenesis and vasculogenesis in chronic ischemic muscle, there is still no knowledge about the effect of shock waves on acute muscle ischemia. However, patients presenting with acute infarction are a major group in the clinical setting. Shock wave treatment as an adjunct to state-of-the-art revascularization in acute limb ischemia or myocardial infarction could dramatically improve outcome and quality of life if the effects were comparable to those seen in the chronic setting.The present experiments therefore were intended to proof whether shock wave treatment induces angiogenesis in acute ischemia and to evaluate a stable long-term benefit resulting in improvement of blood perfusion.www.nap.edu/catalog/5140.html).The experiments were approved by the institutional animal care and use committee at Innsbruck Medical University and by the Austrian ministry of science (BMWF-66.011/0153-II/3b/2012). The investigation conformed to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health  weighing 25–30 g were randomly divided into 4 groups (n = 10). Treatment animals (SWT) received shock wave therapy immediately after hind limb ischemia induction. Control animals (CTR) were treated equally without receiving treatment. The animals were sacrificed 72 h and 28 days after therapy.Anesthesia was administered by an intraperitoneal injection of ketamine hydrochloride  and xylazine hydrochloride . Left femoral artery was ligated and excised between the inguinal ligament and proximal to the branching into saphenous and popliteal artery using 7-0 polypropylene sutures . For analysis, the whole adductor muscle was harvested and split in half resulting in a proximal and distal portion. Of the distal portion, the middle part around the former femoral artery was used for analysis to avoid sampling variances of regions with greater or lesser ischemia.2 at a frequency of 5 Hz. The overall treatment time was about 3 minutes. The rationale or the treatment parameters is our experience from previous studies Treatment was applied as described elsewhere −ΔCT, where ΔCT was defined as CT (GOI) – CT (β-actin).RNA was isolated from muscle tissue using RNeasy Mini Kit  according to the manufacturers instruction. RNA integrity was evaluated with agarose gel electrophoresis and RNA quantity was determined by spectrophotometry. Thereafter, one microgram of RNA was reverse transcribed using the IScript cDNA Synthesis Kit  and till further use stored at −20°C. All qRT-PCR were carried out on a BioRad iQ5 Cycler  as described previously 5′-GGCTGTATTCCCCTCCATCG-3′, reverse: 5′-CCAGTTGGTAACAATGCCATGT-3′; vascular endothelial growth factor (VEGF) - A, forward: 5′-GCACATAGAGAGAATGAGCTTCC-3′, reverse: 5′- CTCCGCTCTGAACAAGGCT-3′; Placental Growth Factor (PGF), forward: 5′-TCTGCTGGGAACAACTCAACA-3′, reverse: 5′- GTGAGACACCTCATCAGGGTAT-3′; FMS-like tyrosine kinase 1 (Flt1), forward: 5′- TGGCTCTACGACCTTAGACTG-3′, reverse: 5′- CAGGTTTGACTTGTCTGAGGTT-3′ and kinase insert domain protein receptor (KDR), forward: 5′- TTTGGCAAATACAACCCTTCAGA-3′, reverse: 5′- GCAGAAGATACTGTCACCACC-3′.The following primer sequences were found using the Harvard’s PRIMER BANK website: β-actin, forward: For in vivo protein analysis muscle samples were homogenized and processed for Western Blotting as suggested by the manufacturer. Monoclonal anti-VEGF antibodies were purchased from Dako .HUVECs were processed for western blotting as suggested by the manufacturer. Polyclonal anti-VEGF antibody was purchased from Abcam  and analyzed 24 hours after treatment. Polyclonal phoshpo-p44/42 MAPK (ERK1/2) antibody was purchased from Cell Signaling Technology . Analysis was performed 30 minutes after treatment.After written informed consent of patients umbilical cords were obtained from Caesarean sections at the Dept. for Gynaecology for isolation of human umbilical vein endothelial cells (HUVECs). Permission was given from the ethics committee of Innsbruck Medical University (No. UN4435). Isolation was performed as described elsewhere To apply shock waves properly to the cells, the T25 culture flasks were dunked into a water bath Human phospho-RTK (receptor tyrosine kinase) and human angiogenesis array Kits  were used for investigation of receptors and proteins involved in HUVEC signaling after SWT. After stimulation with SWT and incubation for 60 minutes, cells were treated as recommended by the manufacturer for RTK profiler analysis. Angiogenesis profiler was performed according to the manufacturers protocol 24 hours after treatment. In each of the profilers a total of 400 µg protein was used.2<0.5%.For achievement of hypoxic condition HUVECs were placed into a common hypoxic chamber for 16 hours with OFor VEGF receptor 2 inhibition HUVECs were pre-incubated for 1 hour prior to treatment with 100 nM Vandetanib purchased from Selleckchem .−1 type I rat tail collagen  in 96 well plates. Aortic rings were observed over a period of 7 days. After fixation with 4% paraformaldehyde aortic rings were stained using Rhodamine labeled Griffonia (Bandeiraea) Simplicifolia Lectin I . DAPI  was used for nuclear counterstaining. Number of rings per group was 6 to 8.The aortic ring assay was performed as described Immunofluorescence staining was performed as described previously Blood flow measurements were performed pre hind-limb ischemia, directly (day 0), 2 and 4 weeks afterwards by a laser Doppler perfusion image analyzer  as previously reported Necrosis score was assessed as described previously All results are expressed as mean ± SEM (standard error of the mean). Statistical comparisons between 2 groups were performed by student’s t-test. Continuous variables were either compared with analysis of variance (Bonferroni) after testing for normality of distribution or the Mann–Whitney test. P-values <.05 were considered statistically significant.+B), as well as of their receptors Flt-1  and KDR  (+D) at 72 hours after left femoral artery excision and shock wave treatment.Real-time PCR analyses revealed higher gene expression of angiogenic growth factors VEGF-A  and PlGF   72 hours after shock wave treatment compared to untreated controls  using receptor tyrosine kinase (RTK) profiler assays. This assay reveals activated receptors by means of phosphorylation. Quantification of relative VEGFR phosphorylation showed a significant, nearly five-fold activation of VEGF receptor 2 (VEGFR) 60 minutes after treatment whereas no effect could be observed on VEGF receptor 1  (C).This indicates a direct effect of SWT on phosphorylation of VEGFR 2, which is known to be the pivotal VEGF receptor for induction of angiogenesis. This confirms our findings of increased numbers of vessels. Thereby phosphorylation of VEGFR 2 may represent the main mechanism of action of shock waves to ischemic muscle. However, treated HUVECs were under common normoxic cell culture conditions. Therefore, we additionally performed Western blots of VEGF in HUVECs that were incubated under hypoxic conditions. Shock wave treated cells showed an increased amount of VEGF protein compared to untreated controls and confirming the findings of in vivo ischemia  (+F). These data indicate that growth factors may mechanically get released from extracellular matrix where they are bound to heparansulfate proteoglycans. However, further experiments are necessary to proof this hypothesis.To further elucidate whether shock waves do as well have a direct effect on growth factor release from endothelial cells we performed an angiogenesis array. It confirmed an increase of VEGF and PlGF protein expression in SW treated endothelial cells compared to untreated controls  (+B). We therefore propose that the increase in microvascular density in ischemic muscles is mainly due to an activation of endothelium. To make sure that sprouts seen under the light microscope actually represent capillaries we performed lectin staining for endothelial cells. Thereby we have been able to show that shock wave induced sprouts from aortic rings indeed are formed by endothelial cells  (+B).Quantitative immunofluorescence was performed to evaluate vessel formation 4 weeks after ischemia and shock wave treatment. It revealed an increased number of CD31 positive capillaries per high-power field , being a correlate for a higher micro-vascular density in the shock wave group compared to controls (+D). This finding 4 weeks after shock wave treatment indicates a stable long-term result of angiogenesis in ischemic tissue.A co-staining for CD31  and alpha smooth muscle actin (staining for smooth muscle cells) was performed to evaluate whether shock wave treatment also induces the formation of mature arterioles that in contrast to capillaries are covered by a smooth muscle layer. Significantly higher numbers of arterioles per high power field could be detected in the SWT group compared to untreated controls  (+B). Although untreated control animals show a notable self-regeneration potential, significant improvement could be observed after 4 weeks in the treatment group   as well as 2 and 4 weeks after femoral artery excision and shock wave treatment (Improvement of blood perfusion as well as of limb necrosis 4 weeks after shock wave treatment shows the clinical benefit of this new treatment option for acute muscle ischemia.Shock wave therapy is developing for a large variety of indications, including acute and chronic soft tissue wounds The present experiments were intended to proof the induction of angiogenesis in the treatment of acute hind-limb ischemia and to evaluate a stable long-term benefit resulting in improvement of blood perfusion.Shock wave treatment as an adjunct to state-of-the-art revascularization in acute myocardial infarction or limb ischemia could dramatically improve outcome and quality of life if the effects were comparable to those seen in the chronic setting.We therefore performed a hind limb ischemia model in mice by excision of the left femoral artery. Changes in gene expression of the main angiogenic factors were measured 72 hours after shock wave treatment. Comparable to results from the treatment of chronic myocardium and muscle ischemia we found an up-regulation of pivotal growth factors VEGF and PlGF mRNA As an underlying mechanism of action of shock wave treatment we revealed a direct effect to VEGF receptor 2 phosphorylation in endothelial cells. Using a VEGFR 2 inhibitor effects on VEGF signaling were almost completely abolished. This confirmed that the shock wave mechanism is VEGFR2 dependent. VEGFR 2 is known to be the pivotal VEGF receptor for the induction of angiogenesis. Indeed, as a consequence we found increased levels of VEGF and PlGF protein after shock wave treatment in endothelial cells. In order to clarify whether endothelial cells may be the main source of angiogenesis induction following SWT we performed an aortic ring assay. It revealed that the induction of capillary sprouting from mouse aorta ex-vivo is possible. Thereby this experiment proofs that myocytes are not a prerequisite source of angiogenesis induction.In line with these findings quantitative immunofluorescence from muscle samples revealed significantly higher numbers of capillaries as well as arterioles covered with smooth muscle cells in treated ischemic muscle compared to untreated controls 4 weeks after induction of ischemia.This higher micro-vascular density may be the main reason for the restoration of blood perfusion as shown by laser Doppler perfusion imaging. Increased blood perfusion resulted in less necrosis in the treatment group.Still today results of surgical and interventional revascularization in limb or myocardial ischemia are limited by non-viable and hibernating myocardium or skeletal muscle due to diffuse artery disease of small vessels Therefore, therapies that induce angiogenesis and muscle regeneration adjunctive to state-of-the-art revascularization might improve survival and functional outcome in patients suffering from acute ischemia. Numerous modalities, including gene and cell-based approaches are currently tested in this respect Biological induction of neovascularization in addition to surgical or interventional revascularization could dramatically improve outcome by recruitment of larger areas of ischemic tissue, such as hibernating myocardium The results of the present study therefore suggest that low energy shock wave treatment could develop a feasible adjunct to surgical and interventional revascularization in the setting of acute myocardial or limb ischemia. The translation of our findings to human application seems close as the treatment of soft tissue wounds already is clinically implemented


Pomacea canaliculata is expanding the rat lungworm disease beyond its native range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on the rat small intestine morphology and physiology.The spread of the invasive snail Through a combination of biochemical, histochemical, histopathological, scanning electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate, hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins, as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days.Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of predators of apple snail eggs. Pomacea canaliculata –known as the apple snail– which have hardly any predators. This freshwater snail is a serious aquatic crop pest in several continents, listed among the 100 worst invasive species. It is the host of a roundworm responsible for the rat lungworm disease causing human eosinophilic meningitis. The spread of this emerging infectious disease has been associated with the expansion of apple snails. They lay eggs above water level in bright pink-reddish masses, presumably a warning coloration. Indeed, eggs have chemical defenses, including neurotoxic and antinutritive proteins. The authors found that the ingestion of egg extracts adversely affects rat small intestine inducing large, reversible changes in the intestinal wall that limits the ability to absorb egg nutrients causing a diminished growth rate. Apple snail eggs are the first animal known to deter predators by this mechanism, but remarkably this defense is rather similar to the toxic effect of plant seeds proteins. These overlapping egg defenses that predators have not managed to overcome yet may partially explain the reproductive success of P. canaliculata.Filled with nutritious substances to nourish the embryos, eggs of most animals are often the targets of pathogens and predators. An exception are the eggs of  Pomacea canaliculata   has become a serious aquatic crop pest in Asia and a vector of the rat lungworm Angiostrongylus cantonensis that causes human eosinophilic meningitis, a potentially fatal disease considered an emerging infectious disease. Unfortunately angiostrongyliasis (rat lungworm disease) continues to be reported in new regions beyond its native range which has been associated with the expansion of this snail P. canaliculata is the only freshwater snail listed among the 100 worst invasive species worldwide P. canaliculata deposit hundreds of bright pink-reddish egg masses, each containing 30–300 eggs Solenopsis geminata . The presence of these egg defenses Rostrhamus sociabilis  and Norway rat Rattus norvegicus  that invariably discard the gland that synthesizes the egg defenses when predating on adult female P. canaliculataThe invasive apple snail P. canaliculata PVF identified a small amount of over 50 other proteins, including two F-type lectins, many proteins involved in innate immunity in other mollusks and some with potential roles against insects and fungi Work in the last two decades identified the perivitellins PcOvo and PcPV2 in the egg defenses against predation As the epithelial cells along the digestive tract of animals are fully exposed to food contents, they are possible target sites for defense proteins. In this regard, plants have evolved a wide array of toxic dietary lectins that interact with the membrane glycoproteins of the luminal side of the gut of higher animals having an important role in plant defenses against predation P. canaliculata PVF on the small intestine of rats. Through a combination of biochemical, histopathological, cell culture and feeding experiments, we provide evidence that oral administration of apple snail PVF adversely affects rat small intestine metabolism and morphology and consequently rat growth rate, presumably by proteins displaying lectin-like activity. This overall effect has not been found in other animals, but it is remarkably similar to that for plant seed lectins on the gastrointestinal tract of rats and other vertebrates.With the aim to further understand the role of egg defenses of a host of the lungworm disease, in the present work we studied the effect of All studies performed with animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals P. canaliculata were collected either from females raised in our laboratory or taken from the wild in streams or ponds near La Plata city, Province of Buenos Aires, Argentina, between November and March of consecutive reproductive seasons. Only egg masses with embryos developed to no more than the morula stage were employed. Embryo development was checked microscopically in each egg mass as described elsewhere Egg masses of All experiments with rats were performed using male Wistar rats from the Animal Facility of the School of Medicine of the National University of La Plata (UNLP), Argentina. Rats came from a colony started with the strain WKAHlHok . Six-week-old animals weighing 180±2 g at the start of the experiments were housed in cages with 12 h day-night cycle, temperature of 22±1°C and relative humidity of 45–60%.et al. Fertilized eggs were repeatedly rinsed with ice cold 20 mM Tris-HCl, pH 6.8, containing a protease inhibitor cocktail  and homogenised in a Potter type homogeniser . Ratio of buffer: sample was kept 5∶1 v/w. The crude homogenates were then sonicated for 15 sec and centrifuged sequentially at 10,000×g for 30 min and at 100,000×g for 60 min. The pellet was discarded and the supernatant comprising the egg PVF was equilibrated in 50 mM phosphate buffer pH 7.4 using a centrifugal filter device of 50 kDa molecular weight cut off  to eliminate potentially interfering compounds. Total protein concentration of the PVF (13.3 g/L) was measured by the method of Lowry et al. PcOvo was purified from the PVF of the eggs by high performance liquid chromatography (HPLC) . First, the sample was analyzed in a Mono Q HR 10/10  using a gradient 0 to1 M NaCl in a 20 mM Tris buffer. The PcOvo peak was then further purified by size exclusion chromatography  using an isocratic gradient of sodium phosphate buffer 50 mM, 150 mM NaCl, pH 7.6. Purity of the single peak obtained was checked by native PAGE performed in a Mini-Protean III System . Protein content was determined by the method of Lowry Effect of egg PVF-supplemented diet on rat growth. Groups of control and treated rats, 10 animals each, were fed ad libitum with a commercial diet for up to 7 days. Treated animals were administered PVF fraction (4 mg protein) in the water on a daily basis, while the control group received the equivalent volume of buffer. Food consumption as well as body weight were determined on a daily basis for each animal. The standard growth rate (SGR) was calculated as follows:to is the initial weight, Wt is the final weight, and t is the time in days. Effect of egg PVF-supplemented diets on rat intestine size and morphology. To establish the time-dependent changes induced by PVF on intestine, rats (groups of 5 rats each) were given either the commercial diet or the diet+PVF feed which contained 4 mg PVF protein rat−1 day−1 and sacrificed after, 4 or 10 days.−1 day−1. Animals were sacrificed after 4 and 8 days and the absorptive surface of the intestine measured as described below (next section).A similar setting was employed using PcOvo-supplemented diet at 4 mg PcOvo protein rat2 inhalation in a closed chamber Rats were euthanized by COSections of the first part of the duodenum were cut, washed several times with PBS to remove food and fixed in 4% formalin in phosphate buffered (pH = 7) for histological examination. In rats from day-4 treatment, sections for SEM analysis were also obtained (see scanning electron microscopy section).−1 day−1. Animals were fed with the extracts for 10 days and then switched to a control diet and sacrificed 4 days after switching treated animals to control feed and processed as above.To establish the time needed to recover after the changes induced by egg PVF on intestine, rats (groups of 5) were given either the commercial diet or a diet-and-egg PVF, which contained 4 mg protein ratCylindrical tissue samples of the small intestine were post fixed in 10% neutral formaldehyde for 24 h at room temperature and then embedded in paraffin wax. Representative 5–7 µm sections were stained with haematoxylin and eosin for histological examination of general morphology. In addition, periodic acid Schiff (PAS) staining was performed to highlight carbohydrate distribution and goblet cells.M was calculated considering these 3 variables, as follows:Fifty properly oriented villi and crypts from duodenum were selected at random from each animal and their length and width measured to calculate mucosal absorptive surface area following the method of Kisielinsky Small intestine sections were assayed by immunohistochemistry (IHC) to evaluate cellular proliferation using a primary monoclonal mouse against the proliferating cellular nuclear antigen (PCNA) as a proliferation marker . The antibody was diluted in 0.1% BSA in phosphate buffer and incubated overnight at 4°C. PCNA is a nuclear acid protein which functions as δ DNA polymerase helper. In the presence of PCNA and a replication C factor, δ DNA polymerase starts the synthesis of DNA and the progression of the cellular cycle.2O2. Positively stained cells showed a golden, dark-brown color. All sections were counterstained with Maeyer haematoxilyn before analysis. Primary antibody was replaced by normal mouse antiserum in control sections.Samples were incubated overnight at 4°C as mentioned above, and visualized using the LSAB kit  detection system which is based on a modified labeled avidin-biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecules. In short, after incubation with the appropriate primary antibody, a sequential 10 min incubation with an anti-mouse biotinylated antibody and peroxidase-labelled streptavidin is performed. Then staining is completed by incubation with 3,3′diaminobenzidine tetrahydrochloride (DAB) and HConcanavalia ensiformis), DBA (Dolichos biflorus), SBA (Glycine max), PNA (Arachis hypogaea), RCA-I (Ricinus communis-I), UEA-I (Ulex europaeus-I) and WGA (Triticum vulgaris) to reveal possible changes of the glycosylation pattern.Small intestine sections were assayed with seven lectins  . Positively-stained cells were demonstrated by a dark golden brown coloration. The sections were counterstained with Maeyer haematoxilyn.They were then hydrated, washed in phosphate-buffered saline, and incubated with biotinylated lectins overnight. Then sections were washed with PBS, followed by 10-min incubation with streptavidin-HRP . Finally the bound lectins were visualized by incubation during 4–10 min with a buffered Tris-HCl solution  containing 0.02% 3,3′-diamino-benzidine tetrahydrochloride (DAB) and 0.05% HAfter 2-hour fixation in 2% (v/v) glutharaldehyde, samples were dehydrated in graded series of ethanol. Then ethanol was replaced by liquid carbon dioxide and samples were dried by critical point in a CP-30 . Samples were gold metalized in a JEOL Fine Ion Sputter, JCF-1100. Observations and photomicrographs were obtained with a JEOL JSM 6360 LV SEM  at the Service of Electron Microscopy, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Argentina.Horse, goat, rabbit and rat erythrocytes were obtained from the animal facilities at the University of La Plata (UNLP). Blood samples were obtained by venous puncture and collected in sterile Elsever's solution  . Prior to use, erythrocytes were washed by centrifugation at 1500 g for 10 min in TBS buffer . This procedure was repeated several times until the supernatant remained clear. Hemagglutinating activity was assayed in microtiter U plates  by incubating a two-fold serial dilution of PVF (6 mg/mL) in TBS with 2% erythrocyte suspension in TBS at 37°C for 2 h. Results were expressed as the inverse of the last dilution showing visible hemagglutinating activity by naked eye.2. Culture medium was replaced every 2 days and subcultured by trypsinization when 95% confluent. Passages 60 through 65 were used for the experiments. Prior to each experiment, the viability of the cells was determined by trypan blue exclusion. Viability of every cell preparation exceeded 90% as determined by counting the stained cells.Human colorectal adenocarcinoma cells (Caco-2) were cultured in Dulbecco's modified Eagle's medium (DMEM) (4.5 g/liter D-glucose) supplemented with 10% newborn calf serum, penicillin (10 U/mL), streptomycin (10 µg/mL), amino acids and vitamins (Life Technologies-Invitrogen). Cells were cultured at 37°C in a humidified atmosphere of 5% COThe cytotoxic effect of the PVF on Caco-2 cells was evaluated using the 3--2,5-diphenyl tetrazolium bromide (MTT) assay %Viability  =  (OD treated cells/OD control cells) ×100Data collected from all experiments were analyzed individually by either t test (histology) or ANOVA (bioassays) using Instat v.3.05 (Graphpad Software Inc.). Where significant differences between samples occurred, a post-hoc Tukey's HSD test was performed to identify the differing means. Results were considered significant at the 5% level.GenBank accession numbers for PcOvo subunits: JQ818215, JQ818216 and JQ818217; GenBank accession numbers for PcPV2 subunits: JX155861 and JX155862.During the first 3 days of treatment with PVF, treated rats showed a significantly lower standard growth rate than the control ones . This efOral administration of PVF for 10 days increased the mean intestinal length of the rats though a tendency was already evident after a 4-day treatment . Within At day 4, samples from control animals showed the characteristic tall, finger-like villi, whereas villi from treated animals showed significantly less height and were wider with some proliferation in the basal zone of the epithelia. In certain areas of the epithelium of treated animals, altered villi with a double, fused or “tongue” shape, displaying a bridge pattern were observed by SEM and light microscopy . PAS staSEM analysis of control and treated animals confirmed the remarkable differences on the length and width of the villi of treated animals . The incPCNA labeling showed moderate immunostaining in the basal areas of the epithelium from controls, while a stronger staining was evident on the treated animals. . Apple snails seem to have evolved passive defense systems to protect their developing embryos; the preferential accumulation of large quantities of lectins, and protease inhibitors is certainly indicative of that strategy. Moreover, it is believed that the main antinutrients responsible for reducing the nutritional value of many plant seeds are a combination of lectin and trypsin inhibitors In this work we demonstrate that the oral administration of apple snail egg PVF promotes alterations in rat growth rate and small intestine morphophysiology for short periods, whereas prolonged exposure to the toxic PVF induces an adaptation overcoming the antinutritonal effects. This defense has not been reported in animals before, but resembles those well established for plant seeds.The severe effects of PVF on digestive tract adds another line of defense to the previously reported suite of biochemical defenses of apple snail eggs. This study helps to explain the near absence of predators and their successful establishment in invaded areas.


Objective. To evaluate the evidence on the immunomodulatory effect of acupoint application for childhood asthma. Methods. Five electronic databases through October 2014 were searched. The risk of bias in eligible studies was assessed using the Cochrane Collaboration tool. Standardised mean difference (SMD) and 95% confidence intervals (CI) of random-effects model were calculated. And heterogeneity was assessed using the Cochran Q statistic and quantified with the I2 index. Results. Six studies were included in our review. The aggregated results suggested that acupoint application showed the beneficial effect for childhood asthma in improving IgA , IgE , IgG , IL-4 , and IFN-γ  but not IgM . And the effective dose of acupoint application may be 2–6 hours/time and a total of 3 times within 4 weeks. Conclusions. This review showed the positive evidence that acupoint application had the favorable immunomodulatory effect for childhood asthma. However, more studies with long follow-up are warrant to confirm the current findings. Asthma is one of the most common chronic respiratory diseases among children, which imposes a substantial care burden on families and health services –3. In UnCommon starting medications for childhood asthma are effective such as short-acting beta2-receptor agonists and inhaled corticosteroids, but adverse drug reactions are still considerable . InhaledCortex Cinnamomi,” Ding Xiang “Flos Caryophylli,” Wu Zhu Yu “Fructus Evodiae,” etc.) with ginger juice [Acupoint application, as a complementary and alternative treatment for asthma, is a kind of herbal cone cake fixed into acupoints . The herbal cone cake is mixed with herbal powders  study design was randomized controlled trials (RCTs); (2) the target population was children diagnosed with asthma; (3) the main intervention should be acupoint application compared with any comparator without acupoint application; (4) the immune outcomes included immunoglobulin, interleukin, and interferon; (5) the study was available in either English or Chinese.Identified abstracts were screened independently by two authors. All full-texts of potentially relevant abstracts were evaluated based on eligibility criteria by one author and confirmed by the other author.Data extraction was performed by two authors independently. They extracted information on the characteristics of study and population, immune outcomes, the follow-up period, and dose of interventions. The corresponding author was contacted when relevant information was not reported. And disagreements were resolved by consensus among authors.The risk of bias in eligible studies was assessed independently by two authors using the Cochrane Collaboration tool , 20. SixQ statistic  and quantified with the I2 index (considered high when I2 was above 75%). Trials, including 2 similar control groups, had the groups combined with the formula provided by the Cochrane handbook for a single pairwise comparison. All meta-analyses were conducted using Cochrane Collaboration software (Review Manager Version 5.2).For continuous data, standardized mean difference (SMD) and 95% confidence intervals (CI) of random-effects model were calculated for all eligible trials. Heterogeneity was assessed with the Cochran n = 18) and not RCTs (n = 4).The process of literature search and study selection was depicted in 6 RCTs , 21–23 oThe risk of bias evaluation is reported in γ (IFN-γ). The results of our meta-analysis suggested that acupoint application showed significant improvements on immune outcomes  should be recommended as a complementary and alternative treatment for childhood asthma, especially in improving immunomodulatory function. And the acupoints usually contain Tiantu “CV 22,” Fei Shu “BL 13,” Dazhui “CV 14,” Gaohuangshu “BL 43,” and so forth.γ was thought to protect against the development of asthma by regulating T helper-2 cytokine production [γ level in patients with asthma [γ may be the basic mechanism of acupoint application for childhood asthma.Assuming that acupoint application was effective for asthma, some complex immunomodulatory mechanisms may provide possible rationales. IgE was considered a potent predictor of the development of asthma and played a central role in the pathophysiology of asthma . Severaloduction . And theh asthma . TherefoSome relevant studies also supported our findings. In the review of herbal interventions for asthma in adults and children, the authors concluded that herbal preparations showed the valuable efficacy for asthma , 40. AndThere were several limitations in our review. First, all eligible studies were conducted in China, which limit the generalizability of our findings due to population characteristics. Second, there were only two eligible trials in some subgroup meta-analyses due to strict eligibility criteria. It may bias aggregated results, but low eligibility criteria would generate more doubtful results. Third, the eligible studies did not focus on lung function of children with asthma because it is difficult to test lung function of children. However, it may affect physicians' decisions of acupoint application for childhood asthma because the improvement of lung function is important for childhood asthma. In addition, serious adverse events were not reported in eligible trials, but it should be careful to conclude that acupoint application was safe because it was not clear whether adverse effects had been measured or not in some studies. These limitations, especially in population characteristics and fewer eligible studies, may affect our findings and restrict the applied range of acupoint application.γ. And the effective dose of acupoint application may be 2–6 hours/time and a total of 3 times within 4 weeks. Considering few included studies in some subgroup meta-analyses and lack of follow-up data, more RCTs with long follow-up are warrant to confirm the current findings.Our review showed the positive evidence that acupoint application had favorable immunomodulatory effects for childhood asthma, especially on IgA, IgE, IgG, IL-4, and IFN-


Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, high-resolution structures with SLS using diverse starting materials , prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 μm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials. The advent of additive manufacturing (AM) technology, also referred to as 3D printing (3DP), has led to its increased use in industry and scientific laboratories, as well as in homes and community makerspaces ,2. In AMSelective Laser Sintering (SLS) is a third and highly versatile AM process that creates solid parts by tracing a laser beam, focused onto a thin layer of powder, in a 2D pattern . The powSLS has gained attention in the bioengineering community in recent years as a platform for the fabrication of scaffolds for bone tissue engineering, with an emphasis on bioactive and bioresorbable materials . ScaffolHere, we describe the development of a low-cost, open-source SLS system (OpenSLS), which makes SLS available to scientists as a highly customizable fabrication platform. We demonstrate the capabilities and limitations of this technology and discuss several possible applications for parts created using OpenSLS. Specifically, we address the production of highly intricate scalable models of physiologic structures, the fabrication of fluidic networks derived from sintered parts, and the implementation of SLS for cell studies aimed at biomaterials science and tissue engineering. We present these diverse applications together to highlight the multifaceted impact that access to low-cost, customizable SLS technology could have on scientific research in various disciplines.We begin by discussing the design and construction of OpenSLS, which harnesses the features of a commercial laser cutter alongside a simple, custom-built powder distribution module. We first establish fabrication of structures with sub-millimeter features and overhanging regions in nylon, a commonly used material in commercial SLS systems. Subsequently, we demonstrate fabrication of PCL into a diamond lattice structure which could be relevant as a model scaffold geometry for bone tissue engineering. We characterize the accuracy and precision of OpenSLS as well as the surface quality and mechanics of sintered structures and show that the fidelity and quality of sintered parts match those of commercially sintered parts. We also introduce a simple post-processing technique to reduce the surface roughness of sintered PCL structures, with a concomitant increase in elastic modulus and yield stress of these scaffolds. Processed PCL structures are also useful for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) (PDMS) silicone. Finally, we demonstrate that human mesenchymal stem cells seeded onto sintered PCL surfaces were viable and able to undergo osteogenic differentiation and matrix mineralization, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for tissue engineering studies.We believe that OpenSLS could serve the scientific community as an accessible platform for fabrication of structures composed of a wide range of materials, including those not supported by commercial SLS suppliers or their maintenance contracts. OpenSLS represents a meaningful addition to the growing body of open-source technologies that give engineers and scientists access to and unprecedented control over advanced fabrication and analytical tools at low cost.Selective Laser Sintering was investigated using nylon, a common industrial SLS substrate, and polycaprolactone (PCL). Nylon-12  has a melting point of 181°C, melt-flow index 50 g/10 min, and reported mean particle size 55 μm. PCL  has a melting point of 58–60°C, melt-flow index 5.2–11.3 g/10 min, and reported particle size below 600 μm. Materials were used as received from the suppliers.2 laser cutter with 60 × 90 cm bed  was selected as the base of the OpenSLS system due to its high power range, modular electronics, and large working envelope. A RepRap Arduino Mega Board , an integrated 3D printer motherboard, was chosen to control OpenSLS due its 5 stepper motor axes, Pulse Width Modulation (PWM) output pins, and compatibility with the widely used open-source Marlin firmware. The laser cutter’s native stepper motors and microstepping drivers were retained for driving its XY gantry, but the motor control lines were re-mapped to motor extension pins on the RAMBo  is designed for extrusion printing, not SLS, so melt extrusion functionalities that are meaningless in an SLS system were removed . Then, G-code commands were assigned for functionalities unique to SLS  and 30 W cm-2 (subsequent layers) while PCL prints used approximately 150 W cm-2 (first 1–3 layers) and 100 W cm-2 (subsequent layers). Higher power settings for the initial layers helped to ensure adhesion between the print and a layer of painter’s tape which covered the build platform; this adhesion prevented warping throughout fabrication process. The initial powder layer was set by manually creating the thinnest possible uniform coating of powder on the build platform while subsequent layers were created automatically by the powder distributor. Following fabrication, excess powder was removed from sintered parts with compressed air or by tapping the part against the benchtop.3D model files were designed and prepared for OpenSLS using a toolchain of open-source software applications developed within the hobbyist maker community. 3D model stereolithography (STL) files were created using OpenSCAD software (openscad.org) or downloaded from an online collection of 3D models (thingiverse.com) The diagrid model and diamond lattice model were listed, respectively, as “Diagrid bracelet” by nervoussystem (n-e-r-v-o-u-s.com), and “Diamond Lattice Model” by pmoews. The reduced diamond lattice was generated by modifying the diamond lattice model in OpenSCAD. Models were sliced into 2D layers as machine-interpretable G-code using Slic3r  was calculated using the formula Scanning electron microscopy (SEM) was used to analyze nylon particle size distribution (prior to sintering) as well as for surface analysis of sintered nylon and PCL structures. For particle sizing, sparsely arranged nylon particles were sputter coated with approximately 10 nm gold  and imaged on an FEI Quanta 400 Environmental Scanning Electron Microscope . PCL particles were too large to be efficiently sized via SEM and were instead sized using an optical stereo microscope  after validating that the sizing was consistent with SEM . For anaMicrocomputed tomography (μCT) was used to analyze the internal structure of sintered parts. Samples were scanned in 0.5 degree steps at 20 μm resolution on a SkyScan 1272 X-ray microCT scanner  equipped with 11 megapixel detector. The X-ray source was set to 50 kV and 200 μA with an exposure duration of 424 ms. Image reconstruction was performed in nRecon software (Bruker) and volumetric visualization was performed in CTVox software (Bruker). μCT data is presented as virtual cross-sections through the volumetric scan, performed in CTVox software.Sintered PCL structures were suspended from the rubber stopper of a 4 L filter flask. Dichloromethane  was added to a round-bottom flask with stopcock and the stopcock was connected to the vacuum adapter on the filter flask. DCM was heated until boiling, then the stopcock was opened to allow DCM vapor to diffuse into the filter flask. The suspended PCL structure was monitored and removed after it appeared sufficiently smoothed . Overexposure to DCM vapor led to deformation of PCL structures due to gravity. Segregation of the liquid DCM in a separate chamber from the specimen was critical for achieving uniform smoothing of exterior and interior surfaces.Two geometries were used to assess dimensional accuracy and precision of nylon and PCL structures. The first was a 10×10 mm cube with 1×1 mm rectangular studs on each face. The 10mm and 1mm features were measured with calipers for 10 samples produced over 3 sintering runs. The second geometry was the full (nylon) or reduced (PCL) diamond lattice model. Lattices were μCT scanned as described above, reconstructed, and exported as STL files. 3D scans were aligned with their corresponding models using Blender (blender.org) and Geomagic Control . Aligned scans and models were divided into corresponding 100μm slices using Creation Workshop (envisionlabs.net). A custom MATLAB script was used to quantify overlapping and non-overlapping pixels in each slice for each scan/model pair.Uniaxial compression testing was conducted in accordance with a modified version of ASTM standard D695-02a. Test samples were cylinders 12.7 mm in diameter and 25.4 mm in height with a 3D rectilinear macroporous network geometry as described by Eshraghi and Das . SamplesSintered PCL structures were vapor-smoothed in DCM and embedded in poly(dimethyl siloxane) . PDMS (10:1 PDMS base to curing agent) was allowed to cure for 48 hours in a 3D-printed container made from poly(lactic acid) (PLA). After curing, the container and PDMS-embedded PCL were stirred overnight in DCM to dissolve the PLA and PCL. The resulting structure was rinsed with 99% isopropyl alcohol (IPA), then soaked in an IPA bath. The internal void space generated by the sintered PCL was visualized by perfusion with a solution of food coloring dye in IPA and by μCT scan.2 in DMEM  supplemented with 10% fetal bovine serum  and 1% antibiotic . PCL platforms (approximately 8×5×2 mm) were sintered and sterilized in 70% ethanol. Half of the PCL platforms were vapor-smoothed in DCM prior to sterilizing and seeding. Labeled hMSCs (passage 5) were seeded on the platforms at a density of 3×106 cells cm-2 in an 80 μL droplet of media. For imaging on an inverted microscope, the platforms were inverted in 24-well plate wells. hMSCs on the platforms were imaged at 3, 7, and 10 days on a Nikon Eclipse Ti inverted epifluorescent microscope (Nikon USA) equipped with a Zyla 4.2 sCMOS camera . Images were acquired in z-stacks to compensate for the uneven surface of the PCL, then focus-stacked using open-source Enfuse software (enblend.sourceforge.net). ImageJ was used to remove background autofluorescence from the PCL and to perform gamma correction to improve cell visualization.Human Mesenchymal Stem Cells  were stably transduced at passage 1 with a second-generation lentivirus in accordance with Rice University Institutional Biosafety Committee oversight on Protocol #662023. Methods for lentiviral transduction were modified from Kutner and colleagues . Lentivi5 cells cm-2 as described above. After 1 day in culture, the platforms were rinsed in PBS and live/dead staining was performed in accordance with the manufacturer’s instructions . PCL boats treated with 70% ethanol prior to staining were used as negative controls. Fluorescent imaging was performed as described above, using exposure times determined by imaging live and ethanol-treated hMSCs on tissue culture plastic. Live and dead cells were counted either manually or using ImageJ and results encompassed three PCL platforms seeded in separate experiments.Unlabeled hMSCs (passage 4–6) were seeded on vapor-smoothed PCL platforms at a density of 3×10Unlabeled hMSCs (passage 2) were seeded on sintered PCL platforms (unsmoothed and vapor-smoothed) as described above. After 1 week incubation in growth media, half of the platforms were changed to osteogenic media, consisting of growth media supplemented with 10 mM β-glycerophosphate and 50 μg/mL L-ascorbic acid (Sigma). The remaining platforms continues to be incubated in normal growth media. 32 days after the switch to osteogenic media, Alizarin Red S staining was performed using a modified protocol from Madurantakam and colleagues . Briefly2 laser cutters use the same wavelength laser (10.6 μm) as commercial SLS systems and are capable of positioning a fine laser spot (approximately 375 μm in our system) with similar spatial precision. While conventional systems use optics to raster scan a laser beam across the powder bed, the laser in OpenSLS travels along a toolpath corresponding to the geometry being sintered.The lack of affordable laser sintering technology for a laboratory setting motivated the construction of an inexpensive open-source Selective Laser Sintering (OpenSLS) system repurposed from a commercial laser cutter. The native functionality of the laser cutter, in combination with a custom-designed powder handling module, creates the fundamental hardware necessary for SLS  with open-source firmware. A wiring diagram for OpenSLS is provided Fig A in , as is tWe laser sintered powdered nylon to validate the ability of OpenSLS to consistently produce solid parts with high resolution and to evaluate the system’s performance . A diamo-2 in nylon). Importantly, the power and speed settings, as well as the layer height, need to be independently optimized for different materials.The parameter space within OpenSLS includes the material, powder layer height, laser speed, and laser power. Optimizing the last two parameters was the primary challenge in achieving consistent sintering with high feature resolution. When the power is too high, over-sintering may occur such that material lying outside of the laser toolpath is fused along with the intended pattern. Additionally, excessive fusion between powder layers may lower the resolution along the build axis of the part. Insufficient laser scanning speed may result in an irregular melt pool as the powder is sintered, leading to distortions or cavities which reduce print quality . AlternaFrom gross examination, nylon appeared to sinter with smooth exterior surfaces. Under SEM examination, we observed a smooth core of fused nylon, with a partial layer of unfused particles loosely attached . This suAmongst bioengineers and medical professionals, there is interest in reproducing physiological structures captured via medical imaging techniques –62. For Following the many studies that used commercial SLS systems to fabricate macroporous polycaprolactone (PCL) structures –36,40,45In lieu of powder size reduction, we developed a surface smoothing technique which uses dichloromethane (DCM) vapor to reduce the surface roughness of sintered PCL . The DCMOpenSLS was evaluated as a manufacturing process through dimensional characterization of sintered parts as well as by mechanical testing. As discussed above, the laser spot size determines the resolution  of sintered parts when small particles such as nylon (~50 μm) are used. Therefore, the dimensional accuracy and precision of nylon  approachThe measured dimensions of PCL are less reflective of the accuracy and precision of OpenSLS because the large particle size (~500 μm) limits the resolution. PCL measured dimensions consistently exceeded their nominal length by 1–2 mm for both centimeter- and millimeter-scale features, which is consistent with our observation that large PCL particles become partially fused at the edges of sintered structures and protrude beyond the nominal dimensions. Unsurprisingly, these results demonstrate poor accuracy compared to previous studies which used more finely milled PCL  and highTo analyze how accurately complex geometries are reproduced in OpenSLS, we performed a dimensional fidelity analysis of nylon and PCL diamond lattices . For nylFor PCL diamond lattices, representative cross-sections highlight the major overprinting that occurs due to PCL grain size . The edga) 34.0 ± 7.6 μm, a value consistent with both the particle size of nylon and prior determinations of Ra for sintered nylon using commercial SLS systems  34Like many additive processes, the time required to build a part using SLS scales with both size and complexity of the 3D model. For example, a solid cube might require less time to pattern than a cube of the same dimensions containing macropores, despite the greater volume of material used in the solid cube. For any part, it is always more efficient to sinter multiple copies at once than single copies sequentially so that the process of creating new layers occurs only once instead of for each model. Similarly, it is least efficient to sinter models whose long axis is oriented in the z-direction because adding new layers is generally more time-intensive than adding geometry to existing layers. Additionally, the efficiency is material dependent; different laser scan speeds are optimal for individual materials, contributing to disparities in build time for the same geometry. For all these reasons, quantifying the efficiency of OpenSLS is difficult and a straightforward relationship between model volume and build time does not exist. In the absence of a predictive relationship to determine build time, the times required to build geometries used in this study are shown .For structures produced via additive manufacturing, mechanical properties vary between geometries and may be markedly different from the bulk material properties. Uniaxial compression testing of nylon and PCL yielded linear stress-strain plots until failure, caused by buckling of vertical filaments . Eshraghet al. measured the compressive modulus for macroporous sintered PCL as 52–68 MPa, in reasonably good agreement with our findings . Wi. Wi36]. findings . The sliMacroporous nylon specimens also underwent compression testing. For nylon, fitting the linear region of the stress-strain curve for 8 samples yielded an average elastic modulus of 45.2 ± 5.6 MPa , yield sAs fluidic networks are increasingly used in microfluidic chips and diagnostic devices, it is valuable to investigate techniques by which such networks can be assembled via additive manufacturing. We used sintered, vapor-smoothed PCL structures to sacrificially template fluidic networks in PDMS. In sacrificial templating, a template structure is fabricated in a temporary material and encased in a second bulk material. Selective removal of the temporary material patterns the template structure as void space within the bulk material ,76. WhenWe also templated a diamond lattice geometry to generate a complex, interconnected fluidic network in PDMS . PerfusiThe biocompatibility of PCL has been validated previously through use in drug delivery devices ,81, tissWe further validated the biocompatibility of sintered PCL platforms through live/dead staining of seeded hMSCs. Over three experiments, we found that approximately 85% of cells were viable 1 day after seeding, demonstrating that losses in viability are minimal . These rCulture of hMSCs on sintered PCL is relevant for bone tissue engineering insofar as the hMSCs can be reliably differentiated along an osteogenic lineage. Indeed, differentiation of hMSC populations into osteoblasts has emerged as a promising strategy for encouraging mineralization and maturation of engineered bone . We veriWe demonstrated an open-source SLS system that can be assembled for under $10,000 USD, representing a 10- to 50-fold decrease in cost compared to commercially available systems. With this system, nylon structures can be fabricated with sub-millimeter features and dramatic overhangs. The potential for sintering relevant materials for tissue engineering was demonstrated through sintering of polycaprolactone, which was compatible with hMSC viability, characteristic morphology, and osteogenic differentiation. OpenSLS could serve the scientific community as an accessible platform for fabrication of structures composed of a wide range of materials, including non-traditional materials not supported by commercial SLS suppliers. Overall, OpenSLS is a powerful and low-cost tool that makes additive manufacturing via laser sintering feasible and cost-effective for scientific laboratories.S1 File(PDF)Click here for additional data file.S1 Bill of Materials(XLSX)Click here for additional data file.S1 Figa,c) SEM micrographs of nylon and PCL powder at 35x demonstrate that the respective raw materials differ in size by approximately an entire order of magnitude . b,d) High-magnification SEM micrographs of nylon and PCL show that both particles have rough surfaces at this scale, but nylon particles are more spherical and less irregular . e) Particle size distributions were determined for nylon using SEM (n = 1007) and for PCL using optical images (n = 1168). Sizing data from optical microscopy was validated by comparing particle measurements with measurements of the same particles using SEM . The siz(TIF)Click here for additional data file.S2 FigThe same sample of PCL was imaged on an optical microscope, then sputter-coated and measured on SEM. Lengths measured with ImageJ from the optical microscope image  were compared to the corresponding length measured on the SEM micrograph . On average, the difference between the size measurements was 3.8%, validating the use of optical microscope images for particle sizing.(TIF)Click here for additional data file.S3 Figa) Rendered CAD model of a macroporous modification to ASTM Standard D695-02a geometry . This modification was introduced previously by Eshraghi and Das, see . b) Modi(TIF)Click here for additional data file.S1 Video(MP4)Click here for additional data file.S2 Video(MP4)Click here for additional data file.


On 10–11 March 2015 University College London hosted the annual Alzheimer’s Research UK Conference. This report provides an overview of the presentations and discussions that took place. The 2015 Alzheimer’s Research UK (ARUK) Conference offered a diverse programme, prepared by a committee led by Sebastian Crutch (University College London (UCL), UK). The programme started with the perspectives of four clinicians, who exposed the singularities and challenges presented by different causes of dementia. These were followed by the latest insights from genetics, pathology and brain imaging.Hugh Markus  showed that patients with small vessel disease, in contrast to patients with Alzheimer’s disease (AD), present a neuropsychological profile with a more prominent deficit of executive function than episodic memory. Frederik Barkhof  described neuroimaging features of patients with vascular dementia and discussed the interplay between vascular and AD pathologies, presenting a correlation between atherosclerotic calcification, cognition and structural brain changes.From work carried out in collaboration with Southampton researchers, John Hardy  suggested that seeding of amyloid-beta (Aβ) plaques and propagation of tau could be triggered by loss of homoeostasis due to age-related failure of lymphatic drainage along walls of aged cerebral arteries. James Nicoll  indicated that, in cerebral amyloid angiopathy (CAA), Aβ accumulation in blood vessel walls disrupted smooth muscle cells, which normally regulate local blood flow to meet neuronal metabolic demand. Nicoll also described seminal work on the analysis of neuropathology cases following AN1792 active immunisation. Whilst amyloid clearance was achieved, increased CAA and CAA-associated vasculopathy and amyloid-related imaging abnormalities (ARIA) suggested that plaques were removed from the brain by vascular routes. A reduction of plaque-associated apolipoprotein E (ApoE) and an increase of vascular ApoE in vessels were also shown. Nick Fox  showed an IgG-titre dose–response whole brain tissue loss following the AN1792 and bapineuzamab trials, including ventricular enlargement. However, Fox argued that, in the bapineuzamab passive immunisation trial, these volume changes were driven by ARIA.Karen Horsburgh  described work in mice with cilostazol, a phosphodiesterase inhibitor with beneficial effects on vascular health. Cilostazol improved spatial working memory and white matter function in mice, and dampened microglial proliferation in the corpus callosum after hypoperfusion.MAPT, C9orf72 and GRN). Drawing from neuropathology studies, Manuela Neumann  discussed findings in C9orf72 mutation cases suggesting a close correlation between the anatomical distribution of TDP-43 with the pattern of neurodegeneration and clinical phenotype. In contrast, the distribution of C9orf72 di-peptide repeats and RNA foci was highly consistent among cases, but showed no clinical correlation (age at onset or duration) or association with neurodegeneration. Neumann also suggested a key role for TDP-43 dysfunction as a downstream effect in C9orf72 mutation-associated pathogenesis. Using a newly generated Drosophila model, Alan Stepto , UK) reported that pan-neuronal expression of 64-repeat and 128-repeat, but not eight-repeat, hexanucleotide expansions of C9orf72 resulted in age-related behavioural deficits that correlated with repeat-associated non-ATG translation of di-peptide repeats. Additionally, Stepto observed premature death and impaired locomotor behaviour in flies expressing high levels of 38-repeat RNA. Chris Shaw  revealed that seven missense mutations have been identified in an undisclosed calcium binding protein in familial and sporadic cases of motor neurone disease. Pathological analysis of one case indicated that the protein formed fibrillary and globular inclusions in neurones and glia as well as classical TDP-43 pathology.Building on recent genetic discoveries, Julie Snowden  showed the relationship between the diverse clinical characteristics and the underlying pathologies of frontotemporal dementia (FTD)  and associated genetic mutations  highlighted the ongoing DIAMOND-Lewy programme  which aims to bring together an integrated diagnostic toolkit and a patient management pathway. As part of this study, which is a collaboration with Newcastle University and Durham University, O’Brien described an ongoing systematic review of dementia with Lewy bodies/Parkinson’s disease dementia trials, in which controlled trials show that cholinesterase inhibitors may have beneficial cognitive effects while uncontrolled trials suggest that levodopa could be helpful for motor symptoms. As a potential diagnostic tool, David Klenerman  showed that, despite the presence of β-sheet oligomers in control samples, total β-sheet α-synuclein oligomers increased in size and number in Parkinson’s disease cerebrospinal fluid (CSF) samples.Illustrated by cases from his own practice, Chris Butler  discussed the typical but clinically heterogeneous features of AD and the tools that can be used to detect them. Nick Fox (UCL) showed how imaging techniques are accurate in revealing hippocampal atrophy up to 5 years before the onset of symptoms with an annual reduction of 3–4 % as opposed to 0.3 % in healthy people. Imaging in posterior cortical atrophy showed widespread cortical amyloid deposition but more selective tau distribution, suggesting that tau is more closely linked to hypometabolism and symptomatology. Henrik Zetterberg  showed that, in AD, CSF Aβ42 levels correlated with the presence of amyloid positron emission tomography and with postmortem amyloid cortical plaque load. Zetterberg also presented the C-terminus of the dendritic protein neurogranin as a potential biomarker for synaptic degeneration across neurodegenerative diseases and showed particular correlation with cognitive decline. The 2015 Nobel laureate John O’Keefe  described his seminal work on spatial cells in hippocampal formation, now being translated into AD diagnostic markers. O’Keefe described how the 4 Mountains Test, a topographical memory task, was highly sensitive to focal hippocampal pathology and had high diagnostic sensitivity and specificity in differentiating mild cognitive impairment with and without CSF biomarkers.At a molecular level, James Duce  presented evidence that iron disruption in AD could be modulated by the processing of amyloid precursor protein (APP) on the cell surface whereby the amyloidogenic pathway that requires BACE1 and leads to Aβ generation also causes intraneuronal iron retention and increases vulnerability to reactive oxygen species neurotoxicity; a common concomitant in AD. Rob Andrew  investigated the interactomes of the APP695 and APP751 isoforms to clarify the mechanisms underpinning their differential processing to Aβ. Despite a significant overlap, the role of APP695 in nuclear signalling and mitochondrial functions was consolidated and higher levels were reported at the plasma membrane than for APP751. Bart De Strooper  described the identification of G-protein coupled receptor 3 (GPR3) as a mediator of Aβ generation through interaction with β-arrestin 2. Subsequent work showed that GPR3 modulation, unlike other γ-secretase modulators, did not impact on notch signalling. Human brain samples showed increased GPR3 expression, and evidence from five mouse models showed that lower GPR3 expression reduced plaque burden, soluble Aβ and behavioural deficits.During the session on tau, Karen Duff  showed that rTgTauEC, a model with predominant expression of abnormal tau in the entorhinal cortex, has reduced place and grid cell firing and that firing patterns across days were less specific, coherent and stable compared with control aged mice. Using these mice crossed with APP/PS1 mice, Amy Pooler  found that at 16 months concurrent amyloid deposition in the cortex led to an increase in the speed of tau propagation to distal areas, and an increase in tau-induced neuronal loss. Duff showed data suggesting that tau accumulated on spine-like structures on the axon and was taken up by cells after a lag phase. In-vitro data presented by Tara Spires-Jones  provided further evidence that tau was required for Aβ-induced synapse loss, possibly mediated by caspase 3. Spires-Jones also showed that the plaque size, dystrophic neurites per plaque and the amount of soluble Aβ in synaptoneurosomes were increased in APP/PS1/hTau mice compared with APP/PS1 mice, correlating with data presented by Pooler. It was suggested that amyloid accumulation could trigger neuronal activity and therefore release of tau. In the P301S mouse model, Maria Grazia Spillantini  determined that several proteins were differentially expressed between 1 and 5 months, as tau pathology and neuronal death progressed. Spillantini also showed that astrocytes and astrocyte culture medium from these mice were toxic to neurones, with a reduction of synaptic protein function.Dominic Walsh  kick-started the annual debate ‘Are oligomers the real target?’ arguing for the motion of Aβ oligomers as the toxic species. He suggested that Aβ, acting as an initiator of AD, would not have to show direct correlation with clinical severity. Walsh showed that certain APPTg mice exhibit cognitive deficits prior to appreciable amyloid deposition and benefit from acute treatment with anti-Aβ monoclonal antibodies, and that human brain-derived, non-fibrillar Aβ has been shown to have disease-relevant activity. De Strooper, arguing against the motion, disputed evidence of Aβ toxicity, citing the gap in concentrations and time courses observed in in-vitro models versus the human disease. He further argued that the large variety of receptors reported to bind Aβ and mediate its effects cannot be specific effects of Aβ. The lively debate highlighted important barriers to overcome, including lack of experimental standardisation and integration across laboratories as well as lack of basic knowledge of oligomer biochemistry and the relationship between Aβ and cognitive decline.RAB39B-containing module in the substantia nigra enriched for genes implicated in mitochondrial function, including SNCA, PARKIN and PINK1. Rita Louro Guerreiro  showed pathway and network analyses that identified immune response modules as the most significant in AD. Guerreiro also highlighted the finding that the neuroinflammation positron emission tomography ligand PK11195 was not effective in 30 % of a Caucasian population owing to a polymorphism in the translocator protein, suggesting that integration of genetic findings is essential to improve diagnosis. Tony Wyss-Coray  presented various analyses of human plasma samples from the umbilical cord and from adults aged 20 and 65 which indicated that prominent changes in secreted signalling proteins correlated with ageing, including an increase in inflammatory proteins and a decrease in growth factors. Wyss-Coray also showed that, in aged NOD SCID gamma mice subjected to heterochronic parabiosis, circulatory plasma factors from young humans could ameliorate cognitive deficits, increase hippocampal expression of plasticity-related genes and increase the number of c-fos-expressing neurons in the dentate gyrus. One of the ‘rejuvenation factors’ in young plasma was identified as colony-stimulating factor 2.Keynote speaker Hardy demonstrated how selective vulnerability may, in part, be a consequence of different neurons being close to differing ‘catastrophic cliffs’ dependent on their function. As an example, Hardy described work using transcriptomics across the human brain that identified a Eric Karran (ARUK) provided an update on ARUK’s initiatives and highlighted the launch of the Drug Discovery Alliance. Three Lead Academic Scientists of the Alliance—David Rubinsztein , Giampietro Schiavo  and Chas Bountra —spoke of their plan to establish Drug Discovery Institutes at their respective institutions and to establish a network with academia and industry to facilitate the progression of lead molecules towards the clinic.http://www.brainsfordementiaresearch.org.uk), Dervis Salih  described Mouseac (www.Mouseac.org) and Braineac (www.Braineac.org), Cathie Sudlow  presented the UK Biobank (www.ukbiobank.ac.uk) and Jonathan Schott  presented a range of imaging resources and image analysis software available at UCL (http://cmictig.cs.ucl.ac.uk/research/software).In a session dedicated to resources, Paul Francis  presented Brains for Dementia Research (


Pulse pressure (PP) is a risk factor for cardiovascular disease. It has been reported that ambulatory blood pressure (BP) and nighttime BP parameters are heritable traits. However, the genetic association of pulse pressure and its clinical impact remain undetermined.We conducted a genome-wide association study of PP using ambulatory BP monitoring in young-onset hypertensive patients and found a significant association between nighttime PP and SNP rs897876 (p = 0.009) at chromosome 2p14, which contains the predicted gene FLJ16124. Young-onset hypertension patients carrying TT genotypes at rs897876 had higher nighttime PP than those with CT and CC genotypes . The T risk allele resulted in a cumulative increase in nighttime PP . An independent community-based cohort containing 3325 Taiwanese individuals  was studied to investigate the genetic impact of rs897876 polymorphisms in determining future cardiovascular events. After an average 7.79±0.28 years of follow-up, the TT genotype of rs897876 was independently associated with an increased risk (in a recessive model) of coronary artery disease  and total cardiovascular events , suggesting that the TT genotype of rs897876C, which is associated with nighttime pulse pressure in young-onset hypertension patients, could be a genetic prognostic factor of cardiovascular events in the general cohort.The TT genotype of rs897876C at 2p14 identified in young-onset hypertensive had higher nighttime PP and could be a genetic prognostic factor of cardiovascular events in the general cohort in Taiwan. Hypertension is a leading cause of death, especially in highly industrialized regions, and is considered to be the major risk factor for cardiovascular disease Pulse pressure (PP), the difference between systolic blood pressure and diastolic blood pressure, represents the hemodynamic load on the vasculature and indirectly measures central aortic stiffness. It has been reported that higher PP is associated with left ventricle hypertrophy Ambulatory blood pressure (ABP) monitoring is a validated and accurate method to evaluate blood pressure during a 24-hour period. The correlation between blood pressure (BP) level and target organ damage, cardiovascular disease (CVD) risk, and long-term prognosis is greater via ABP monitoring than official BP measurements. Additionally, PP heritability is higher (0.47–0.57) when using ABP measurements rather than single-point measurements. This indicates that higher heritability is associated with an increased number of BP measurements; therefore, ABP monitoring is more appropriate for the study of the genetics of essential hypertension A two-step genetic association analysis of young-onset hypertension was performed to analyze the genetic association of ambulatory PP in young-onset hypertension. In the first step, a GWAS was conducted with young-onset hypertensive patients as the discovery group (n = 382) to find significant SNP markers. In the second step, a replication study was conducted to test whether SNP markers filtered in first stage were significantly associated with PP in the replication samples (n = 559). The diagnostic criteria for young-onset hypertension have been published previously To determine the clinical implications of a genetic association with pulse pressure found in young-onset hypertension, CardioVascular Disease risk FACtors Two-township Study (CVDFACTS), a cohort study (n = 3325), was selected for investigating the association of future cardiovascular events and the genetic risk variants. The CVDFACTS cohort is a community-based follow-up study begun in 1989 to investigate the cardiovascular disease occurrence and risk factors in Taiwan We calculated the power of our two-stage genetic association study using CaTS software −7), call rates <95%, or minor allele frequency <1%. Genotyping for the verification study and subsequent genotyping of subjects in the CVDFACTS study were conducted using the Sequenom MassARRAY System  by the Academia Sinica National Genotyping Center .Genomic DNA was extracted from peripheral blood samples of hypertensive subjects using the Puregene DNA isolation kit  for the young-onset hypertension genetic study and using the phenol/chloroform method for CVDFACTS. For the GAWS, genotyping experiments were performed with leukocyte DNA using the Illumina Infinium II HumanHap550 SNP chips , which included 560,186 tagging SNPs selected from phase I and II of the HapMap Project. Genotyping assays were performed by deCODE genetics  for 382 young-onset hypertensive subjects in the first stage of the study. We followed the WTCCC criteria for quality control; in brief, individuals were excluded if more than 3% of the genotype data were missing. SNPs were excluded if they showed violation of the Hardy-Weinberg equilibrium  were analyzed separately. To estimate the effect of genetics in determining ambulatory BP, a stepwise linear multiple regression model was used. The genome-wide significance threshold for the p-value was 5×10−7) for the trait of nighttime PP was rs6696698, located on chromosome 1  in the replication study . mosome 1 . Multidimosome 1 . In the To determine the clinical significance of the nighttime PP-associated SNP found among young-onset hypertensive, genotyping for rs897876 was performed in a prospective cohort study, the CVDFACTS study, to evaluate the association of future cardiovascular events with rs897876. The genotype call rate was above 99.4%. The CVDFACTS study enrolled a total of 3325 subjects, 1513 males and 1812 females, with a mean age of 50.2±12.3. The CVDFACTS cohort used in this study consisted of a total of 389 hypertensive patients, 204 (52%) of whom were young-onset hypertensive patients. The young onset hypertensive subjects have higher diastolic blood pressure and higher BMI. Subject with hypertension diagnosed after age 50 have higher fasting glucose, and higher triglyceride values . After aIn our present study, we first identified rs897876 at 2p14, which is highly associated with nighttime pulse pressure in young-onset hypertension. Subjects who carried the TT genotype at rs897876 had higher nighttime PP, indicating that the T allele of rs898786 is an independent predictor associated with higher nighttime PP . Furthermore, the T allele of rs897876 was associated with an increased risk of developing future CAD and total cardiovascular events in an independent cohort. Consequently, the T allele of rs897876 could be a genetic prognostic factor for long-term outcomes in general cohort that include hypertensive patients and could be seen as a genetic marker for advanced cardiovascular events. Furthermore, 2p14 is a locus of interest for further investigation.Recent large-scale GWAS have reported more than 20 novel loci for SBP and DBP where alleles have effect sizes of up to 0.5–1 mm Hg SPRED2 (65M) is a negative regulator of the hypothalamic-pituitary-adrenal axis and contributes to the modulation of hyperaldosteronism and homeostatic imbalances RAB1A (65M) has been reported to be associated with cardiomyopathy 2 = 0.83) as well as pulse pressure (PP) . Further studies will be required to clarify functional relation between FLJ12164 and TGFA responsible for blood pressure regulation. Interestingly, a recent linkage study investigating heritability of PP among Chinese twin pairs found3 linkage peaks on chromosomes 11, 12 and 18 It is interesting that different gene sets seem to contribute to the regulations of daytime and nighttime ambulatory BP. This finding is in accordance with previous findings that the gene sets that regulate daytime and nighttime blood pressure overlap, but there is a genetic component that is specific to the nighttime BP control In our study, the T allele of rs897876 located on 2p14 was independently associated with an increased risk of CVD in a prospective cohort. Currently, accumulating evidence demonstrates that ambulatory BP is a better predictor of morbidity and mortality than conventional BP There are some limitations to our study. There were not many significant associations between individual risk alleles and clinical events in the cohort. This result was expected given that alleles of small effect were tested in a community-based sample of modest size. Although none of the SNP markers achieved genome-wide significance in our first stage GWAS due to small sample size, the second stage replication study still revealed significant loci related to nighttime PP. Further studies with larger sample size and different ancestry groups followed by additional functional confirmation studies are warranted. The second limitation of this study is that ambulatory BP monitoring was not available in the CVDFACTS cohort. The finding that the T allele of rs897876 is associated with nighttime PP was found using ABP monitoring, but the association between 24-hour ambulatory BP parameters among subjects in CVDFACTs could not be evaluated.In conclusion, we first identified rs897876 at 2p14 to be highly associated with nighttime PP in young-onset hypertension patients. Subjects carrying the TT genotype of rs897876 had a higher nighttime PP, suggesting that the T allele of rs897876 was an independent predictor in determining ambulatory nighttime PP. Although the function of this locus is not well understood, the CVDFACTS prospective cohort study clearly demonstrated that the TT genotype at rs897876 on 2p14 is significantly associated with an increased risk of future cardiovascular events. This suggests that rs897876 could be a genetic prognostic factor for cardiovascular events in a general cohort in Taiwan and that 2p14 may have an important role in the pathogenesis of cardiovascular diseases.Figure S1Multidimensional scaling (MDS) analysis plot. The MDS plot shows the first two principal components, estimated by PLINK, based on genotype data from 509,174 SNPs. No population stratification for YOH cases in the first stage was detected .(TIF)Click here for additional data file.Table S1Top ten Eigen vectors of the covariance matrix between the initial stage and the second stage.(DOCX)Click here for additional data file.Table S2Baseline characteristics of patients with essential hypertension.(DOCX)Click here for additional data file.


Administration of low molecular weight heparin (LMWH) is recommended for prophylaxis of venous thromboembolism in patients undergoing hip surgery. In this context, heparin-induced thrombocytopenia (HIT) type II is a complication of rare incidence but sometimes fatal outcome.A 52-year old obese patient undergoing antithrombotic therapy with Enoxaparin after hip surgery presented with a painful, swollen leg and thrombocytopenia on day eight after surgery. Medical history showed previous administration of Enoxaparin without complications 2 years ago. Further diagnostic investigation supplied evidence of multiple thromboembolic events and concomitant compartment syndrome. Administration of Enoxaparin was stopped immediately and treatment with Argatroban was initiated. Diagnosis of HIT was confirmed according to current guidelines. Despite interventional thrombectomy and fasciotomy, amputation of both lower limbs had to be performed due to ongoing necroses. After a 30-days-stay at the intensive care unit because of sepsis, respiratory and renal failure, clinical condition improved and the patient could be transferred for rehabilitation.HIT II is known as complication of administration of LMWH in the perioperative setting. Diagnosis results from clinical findings and platelet count. Argatroban is recommended as an alternative therapeutic anticoagulant in HIT II. Inflammation and surgical trauma are discussed as priming factors to increase risk of HIT II.Administration of LMWH may result in HIT II despite prior uneventful drug exposure. Except for immediate diagnosis, only consequent anticoagulation can stop the course of disease. Hence, interdisciplinary awareness is inevitable for early diagnosis and accurate therapy to prevent from a catastrophic clinical course. Administration of low molecular weight heparin (LMWH) is recommended for prophylaxis of venous thromboembolism (VTE) in patients undergoing hip surgery  of thrombocytopenia whereas if LMWH have been administered more than 3 months ago, the onset of thrombocytopenia seems to be indistinguishable from patients having had their initial exposure to heparin   after hip surgery presented with ongoing pain, a swollen leg and isolated necrotic spots on day eight after surgery. Medical history revealed previous uneventful administration of Enoxaparin 2 years ago. Despite from normal platelet count (286G/l) at the beginning of LMWH therapy, standard laboratory parameters now showed a new onset of thrombocytopenia (33 G/l). Clinical state, results of ultrasound of the lower limb and a CT-angiography supplied evidence of a major thromboembolic event within the right arteria femoralis superficialis and concomitant compartment syndrome. Administration of Enoxaparin was stopped immediately and treatment with Argatroban (Argatra®) was initiated. According to the 4T’s score (8 points) and the detection of PF4-heparin antibodies via an ELISA immunoassay, HIT diagnosis was likely and finally confirmed with heparin-induced platelet aggregation assay (HIPAA).A male 52-year-old patient (body mass index 43 U) undergoing antithrombotic therapy with Enoxaparin  afterwards according to current bridging guidelines. The patient could be transferred to the normal ward and further rehabilitation.Throughout a 30-days stay at the intensive care unit, hemodynamic and respiratory parameters stabilised and the patient regained renal function. Moreover, platelet count normalized ] resulted in a catastrophic clinical course of HIT II. Despite immediate diagnosis and adjustment to recommended therapeutic anticoagulant regime, the progress of the disease led to amputation of both lower limbs. Potential causes of this catastrophic outcome are discussed below.Although drug exposure was uneventful 2 years ago, administration of LMWH [Enoxaparin  antibodies against platelet factor 4 (PF4)/heparin complexes play a major role in development of HIT II. PF4 on the one hand has pro- and anticoagulant function in haemostasis. Antibodies against PF4/heparin complexes generate an activation of platelets with release of procoagulant microparticles and increased generation of thrombin leading to thrombocytopenia based on platelet consumption within thrombotic formations and life-threatening arterial and venous thromboembolic events  comparable to gram negative sepsis  due to prior immune response —a hepatobiliary eliminated direct thrombin inhibitor—is recommended as an alternative anticoagulant for therapy of HIT II (grade 1C). (Linkins et al. In the reported case clinical and laboratory findings led to immediate initiation of evidence based therapy. Following the current ACCP guidelines Argatroban (ArgatraAdministration of LMWH in order to prevent from thromboembolic events can result in HIT II despite prior uneventful drug administration.Except for immediate diagnosis, only consequent anticoagulation can stop the course of the disease. Hence, interdisciplinary awareness is inevitable for early diagnosis and accurate therapy to prevent from a catastrophic clinical course.


We have previously established a mouse model of spontaneous cartilage repair following a full thickness injury and are studying the contribution of mesenchymal stem cells (MSCs) to this process. To determine the contribution of MSCs from bone marrow, we developed a chimeric model by transplanting eGFP+ bone marrow cells into lethally myeloablated wild-type mice. Stromal cell chimerism was confirmed in bone marrow by flow cytometry based on the CD45-/Sca1+/Pdgfr-α+/GFP+ selection and CFU-F assay. GFP+ cells were first detected in synovium at 7 days post-transplant (5%), which increased to 40% of nucleated cells at 8 weeks. GFP+ cells in the synovium were positive for stromal cell markers CD44 and cadherin11, but not endothelial cell markers. To further validate this model, we assessed the response of donor cells to cartilage injury in the synovium post-transplant. Chimeric mice underwent joint surface injury and immediately received a nucleoside analogue chlorodeoxyuridine (CldU) in water for 7 days, after which mice were analysed. There was an increase of donor-derived GFP+ cells in synovium of injured mice compared to uninjured, however, the number of proliferating cells (GFP+/ CldU+) did not increase significantly, suggesting that bone marrow derived cells do not proliferate in situ, but rather infiltrate from bone marrow. In conclusion, we established a chimeric mouse model that will allow us to track contribution of bone marrow cells to cartilage repair.


Dermatophagoides siboney and the combination adjuvant containing a TLR-4 ligand and alum. We propose that IgG1/IgE ratio is effective to assess the clinical efficacy of this experimental vaccine. Aim: To evaluate the immunogenicity of formulation variants of a D. siboney adjuvanted vaccine with varying levels of allergen adsorption on to alum.The new anti allergic vaccine is based on purified allergens from Vaccine formulations with different allergen adsorption levels were obtained by variation of phosphate ions and alum hydroxide content. In a preventative experimental setting, BALB/c mice were treated subcutaneously with 3 doses of each vaccine variant seven days apart. Further, mice were subjected to aerosol allergen challenge. The allergen-specific antibody response was assessed by determining serum levels of IgE, IgG, IgG1 and IgG2a by ELISA, as well the local allergic response by cytokine levels in broncho-alveolar lavage.All variants induced allergen-specific IgG1 and IgG2a antibodies after three immunization doses. The low alum variant showed significant reduction of IgE, whereas the variant with the highest adsorption level (lowest phosphate content) showed significant increase of IgG1. In contrast IgG2a antibodies were not affected by allergen adsorption. A significant increase of the IgG1/IgE ratio was observed for all variants in challenged mice as compared to Th2 controls, with preponderance of the variants with reduced alum content and highest adsorption level. Cytokine levels in BAL indicated a mild but significant shift from Th2 to Th1/Th0 pattern. Histological examination of lungs showed a diminished allergic inflammatory response in vaccinated mice. Reduced size of granulomes at injection site was noted in subjects vaccinated with the low alum formulation variant.Serum IgG1/IgE ratio can be indicative of a protective immunogenicity in a murine model of respiratory allergy. Higher allergen adsorption on to alum could be related to a more effective presentation pathway, or to the function of IgG1 as blocking antibody.