Sustained release of ancillary amounts of testosterone and alendronate from PLGA coated pericard membranes and implants to improve bone healing.

Testosterone and alendronate have been identified as two bone healing compounds which, when combined, synergistically stimulate bone regeneration. This study describes the development of a novel ultrasonic spray coating for sustained release of ancillary amounts of testosterone and alendronate encapsulated in PLGA 5004A as a carrier. Due to the low amounts of testosterone and alendronate used, sensitive in vitro assays were developed to determine in vitro release. The ultrasonic spray coating technology was optimized for coating titanium screws and pericardial collagen membranes, with the aim to improve osseo-integration and (guided) bone regeneration, respectively, without interfering with their primary mode of action. In vitro release analysis of collagen membranes and screws showed up to 21 days sustained release of the compounds without a burst release. Subsequent preclinical studies in rat and rabbit models indicated that testosterone and alendronate coated membranes and screws significantly improved bone regeneration in vivo. Coated membranes significantly improved the formation of new bone in a critical size calvarial defect model in rats (by 160% compared to controls). Coated screws implanted in rabbit femoral condyles significantly improved bone implant contact (69% vs 54% in controls), bone mineral density (121%) and bone volume (119%) up to 1.3 mm from the implant. Based on the results obtained, we suggest that implants or membranes enabled with local sustained delivery of ancillary amounts of testosterone and alendronate can be a promising system to stimulate local bone regeneration resulting in improved osseo-integration of implants and improved healing of bone defects and fractures.

Certainty-based marking in a formative assessment improves student course appreciation but not summative examination scores.

BACKGROUND: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation. METHODS: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs. RESULTS: A total of 389 students participated in the study. After completion 159 (41%) filled in a questionnaire on their experience with CBL during supervised CAFAs. In the control group the median duration of student-instructor interactions was 90 s (range 60-140 s), and this increased with 20 s to 110 s (range 60-150 s) in the group working with a CBL-based CAFA. The number of interactions was similar in both groups (0.22 per student per hour, regardless of CBL inclusion). Forty percent of the students expected that CBL would positively influence their study behavior, and 23% also anticipated a positive effect on examination scores. Student examination scores, however, were not affected by CBL. Almost half of the students (43%) were in favor of CBL inclusion in future computer-assisted learning modules, whereas 33% did not see merit in including CBL in CAFAs. CONCLUSIONS: Incorporation of CBL in a single formative assessment led to a slight increase in student-instructor interaction times, but had effect neither on the number of student-instructor interactions nor on exam scores. CBL inclusion positively influenced student's appreciation of the coursework, presumably by helping students to evaluate their mastery level and identify misconceptions. A more extensive enrollment of CBL beyond an individual formative assessment, throughout a course or a curriculum, may possibly reveal positive effects on study efficacy.

Reversible infertility in a liver receptor homologue-1 (LRH-1)-knockdown mouse model.

Liver receptor homologue-1 (LRH-1) is an orphan nuclear receptor that has been implicated in steroid hormone biosynthesis and fertility. Herein we describe a transgenic inducible short hairpin (sh) RNA mouse model that was used to study the effect of transient LRH-1 knockdown in vivo. Induction of expression of the shRNA directed against LRH-1 for 2-6 weeks resulted in 80% knockdown of LRH-1 protein in the ovary and complete infertility. Gonadotropin hyperstimulation could not rescue the observed defects in ovulation and corpus luteum formation in LRH-1-knockdown mice. The infertility phenotype was fully reversible because LRH-1-knockdown females became pregnant and delivered normal size litters and healthy pups after cessation of LRH-1 shRNA expression. Timed ovarian microarray analysis showed that, in line with the observed decrease in plasma progesterone levels, key steroid biosynthesis genes, namely Star, Cyp11a1, Hsd3b and Scarb1, were downregulated in LRH-1-knockdown ovaries. In contrast with what has been described previously, no clear effect was observed on oestrogenic activity in LRH-1-knockdown mice. Only Sult1e1 and, surprisingly, Hsd17b7 expression was modulated with potentially opposite effects on oestradiol bioavailability. In conclusion, the fully reversible infertility phenotype of LRH-1-knockdown mice shows the feasibility of an LRH-1 antagonist as new contraceptive therapy with a mechanism of action that most prominently affects cholesterol availability and progesterone production.

Accurate gaze direction measurements with free head movement for strabismus angle estimation.

We present the Delft Assessment Instrument for Strabismus in Young children (DAISY) a device designed to measure angles of strabismus in young children fast and accurately. DAISY allows for unrestrained head movements by the mean of a triple camera vision system that simultaneously estimates the head rotation and the eye pose. The device combines two different methods to record bilateral eye position: corneal reflections (Purkinje images) and pupillary images. Detailed results are provided on three orthotropic subjects (age 25-27). Three different conditions were tested: 1) gaze ahead, 2) gaze ahead with different head rotations, and 3) fixed head with different eye positions. Systematic errors occurred between subjects that need further study. The system reached sufficient accuracy to be applied for the measurement of angles of strabismus, almost independent from the head pose.

Gender-specific increase of bone mass by CART peptide treatment is ovary-dependent.

Cocaine- and amphetamine-regulated transcript (CART) has emerged as a neurotransmitter and hormone that has been implicated in many processes including food intake, maintenance of body weight, and reward, but also in the regulation of bone mass. CART-deficient mice are characterized by an osteoporotic phenotype, whereas female transgenic mice overexpressing CART display an increase in bone mass. Here we describe experiments that show that peripheral subcutaneous sustained release of different CART peptide isoforms for a period up to 60 days increased bone mass by 80% in intact mice. CART peptides increased trabecular bone mass, but not cortical bone mass, and the increase was caused by reduced osteoclast activity in combination with normal osteoblast activity. The observed effect on bone was gender-specific, because male mice did not respond to treatment with CART peptides. In addition, male transgenic CART overexpressing mice did not display increased bone mass. Ovariectomy (OVX) completely abolished the increase of bone mass by CART peptides, both in CART peptide-treated wild-type mice and in CART transgenic mice. The effect of CART peptide treatment on trabecular bone was not mediated by 17ß-estradiol (E(2)) because supplementation of OVX mice with E(2) could not rescue the effect of CART peptides on bone. Together, these results indicate that sustained release of CART peptides increases bone mass in a gender-specific way via a yet unknown mechanism that requires the presence of the ovary.

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs.

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

Early postnatal lethality and cardiovascular defects in CXCR7-deficient mice.

CXCR7 is a G-protein coupled receptor that was recently deorphanized and shown to have SDF1 and I-TAC as high affinity ligands. Here we describe the characterization of CXCR7-deficient mice that were generated to further investigate the function of this receptor in vivo. Expression analysis using a LacZ reporter knockin revealed that postnatally Cxcr7 was specifically expressed in cardiomyocytes, vascular endothelial cells of the lung and heart, the cerebral cortex and in osteocytes of the bone. Adult tissues revealed high expression in cardiomyocytes and osteocytes. The observation that 70% of the Cxcr7-/- mice died in the first week after birth coincides with expression of Cxcr7 in vascular endothelial cells and in cardiomyocytes. An important role of CXCR7 in the cardiovascular system was further supported by the observation that hearts of the Cxcr7-/- mice were enlarged, showed myocardial degeneration and fibrosis of postnatal origin, and hyperplasia of embryonic origin. Despite high expression in osteocytes no apparent bone phenotype was observed, neither in combination with ovariectomy nor orchidectomy. Thus as CXCR7 does not seem to play an important role in bone our data indicate an important function of CXCR7 in the cardiovascular system during multiple steps of development.

Identification of secreted cysteine proteases from the parasitic nematode Haemonchus contortus detected by biotinylated inhibitors.

Seven cathepsin B-like cysteine proteases (CBLs) were identified from the immunoprotective excretory-secretory products of Haemonchus contortus. Two-dimensional (2-D) zymography and biotinylated inhibitors were employed to localize active CBLs in 2-D protein gels. Mass spectrometry provided the identification of AC-4, HMCP1, HMCP2, and GCP7 as well as three novel CBLs encoded by clustered expressed sequence tags.

Vaccination-induced protection of lambs against the parasitic nematode Haemonchus contortus correlates with high IgG antibody responses to the LDNF glycan antigen.

Lambs respond to vaccination against bacteria and viruses but have a poor immunological response to nematodes. Here we report that they are protected against the parasitic nematode Haemonchus contortus after vaccination with excretory/secretory (ES) glycoproteins using Alhydrogel as an adjuvant. Lambs immunized with ES in Alhydrogel and challenged with 300 L3 larvae/kg body weight had a reduction in cumulative egg output of 89% and an increased percentage protection of 54% compared with the adjuvant control group. Compared to the adjuvant dimethyl dioctadecyl ammonium bromide, Alhydrogel induced earlier onset and significantly higher ES- specific IgG, IgA, and IgE antibody responses. In all vaccinated groups a substantial proportion of the antibody response was directed against glycan epitopes, irrespective of the adjuvant used. In lambs vaccinated with ES in Alhydrogel but not in any other group a significant increase was found in antibody levels against the GalNAcbeta1,4 (Fucalpha1,3)GlcNAc (fucosylated LacdiNAc, LDNF) antigen, a carbohydrate antigen that is also involved in the host defense against the human parasite Schistosoma mansoni. In lambs the LDNF-specific response increased from the first immunization onward and was significantly higher in protected lambs. In addition, an isotype switch from LDNF-specific IgM to IgG was induced that correlated with protection. These data demonstrate that hyporesponsiveness of lambs to H. contortus can be overcome by vaccination with ES glycoproteins in a strong T-helper 2 type response-inducing aluminum adjuvant. This combination generated high and specific antiglycan antibody responses that may contribute to the vaccination-induced protection.