bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Artificial Insemination Protocol and Dose of Frozen/Thawed Stallion Semen on Pregnancy Results in Mares
Authors: Govaere JLJ , Hoogewijs MK , De Schauwer C , De Vliegher S , Van Soom A , Duchateau L , de Kruif A
Published: April 2014

Contents

Deep intra‐uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra‐uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen–thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi‐flexible Minitube pipette was compared with a newly designed insemination device with a more flexible telescopic insemination catheter (Ghent device). The semi‐flexibleMinitube pipette performed better than the newly designed insemination device with respect to pregnancy outcome (p = 0.008). The superiority of deep horn insemination over uterine body insemination was reflected by the better pregnancy rates obtained after deep insemination using the same low doses (30.6% better pregnancy rates) (p = 0.0123).
Detection of the Matrix Metalloproteinases MMP‐2 and MMP‐9 and Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐2 in Llama (Lama glama) Oviduct
Authors: Zampini R , Argañaraz ME , Miceli DC , Apichela SA
Published: April 2014

Contents

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in several reproductive events like oocyte–spermatozoa interaction and semen liquefaction. In order to study their role in the llama oviductal reproductive process, MMP activity in oviductal fluid (OF) was assayed. Considering that llama genome sequences are partially known, a strategy to procure cDNA sequences of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 was designed. Afterwards, their expression patterns in the different llama oviductal segments were assayed. Gelatine zymograms detected 62 and 94 kDa protease activities that matched MMP‐2 and pro‐MMP‐9, respectively. Expression pattern analysis showed that MMP and TIMP mRNAs were present in ampulla, isthmus, utero‐tubal junction (UTJ) and papilla. Altogether, these findings support the argument that MMPs/TIMPs are produced in the oviduct and secreted into the oviductal lumen. Our results encourage further studies to elucidate the role of these proteins in reproductive oviductal events.
Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae
Authors: Saenz‐de‐Juano MD , Marco‐Jimenez F , Viudes‐de‐Castro MP , Lavara R , Vicente JS
Published: April 2014

Contents

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre‐implantation embryos and its implication in post‐implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post‐transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre‐implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post‐transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.
The Regulation of Aromatase and Androgen Receptor Expression During Gonad Development in Male and Female European Eel
Authors: Peñaranda DS , Mazzeo I , Gallego V , Hildahl J , Nourizadeh‐Lillabadi R , Pérez L , Weltzien F‐A , Asturiano JF
Published: April 2014

Contents

This research investigated the regulation of aromatase and androgen receptor gene expression in the brain–pituitary–gonad (BPG) axis of male and female European eels (Anguilla anguilla) during induced sexual maturation. Complete A. anguilla aromatase (aa‐cyp19a1) and partial androgen receptor α and β (aa‐ara and aa‐arb) sequences were isolated, and qPCR assays were validated and used for quantification of transcript levels for these three genes. Expression levels of the genes varied with sex, tissue and stage of maturation. aa‐arb was expressed at higher levels than aa‐ara in the pituitary and gonad in both sexes, suggesting aa‐arb is the physiologically most important androgen receptor in these tissues. In the female brain, a decrease in aa‐ara and an increase in aa‐cyp19a1 were observed at the vitellogenic stage. In contrast, a progressive increase in all three genes was observed in the pituitary and ovaries throughout gonadal development, with aa‐arb and aa‐cyp19a1 reaching significantly higher levels at the vitellogenic stage. In the male pituitary, a decrease in aa‐arb and an increase in aa‐cyp19a1 were observed at the beginning of spermatogenesis, and thereafter remained low and high, respectively. In the testis, the transcript levels of androgen receptors and aa‐cyp19a1 were higher during the early stages of spermatogenesis and decreased thereafter. These sex‐dependent differences in the regulation of the expression of aa‐ara, aa‐arb and cyp19a1 are discussed in relation to the role of androgens and their potential aromatization in the European eel during gonadal maturation.
Immunohistochemical Localization of Fibroblast Growth Factor‐2 in the Sheep Ovary and its Effects on Pre‐antral Follicle Apoptosis and Development In Vitro
Authors: Santos JMS , Menezes VG , Barberino RS , Macedo TJS , Lins TLB , Gouveia BB , Barros VRP , Santos LP , Gonçalves RJS , Matos MHT
Published: April 2014

Contents

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM+) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.
A Joint Modelling Approach for Longitudinal Studies
Authors: Zhang Weiping , Leng Chenlei , Tang Cheng Yong
Published: April 2014

In longitudinal studies, it is of fundamental importance to understand the dynamics in the mean function, variance function and correlations of the repeated or clustered measurements. For modelling the covariance structure, Cholesky-type decomposition-based approaches have been demonstrated to be effective. However, parsimonious approaches for directly revealing the correlation structure between longitudinal measurements remain less well explored, and existing joint modelling approaches may encounter difficulty in interpreting the covariation structure. We propose a novel joint mean–variance correlation modelling approach for longitudinal studies. By applying hyperspherical co-ordinates, we obtain an unconstrained parameterization for the correlation matrix that automatically guarantees its positive definiteness, and we develop a regression approach to model the correlation matrix of the longitudinal measurements by exploiting the parameterization. The modelling framework proposed is parsimonious, interpretable and flexible for analysing longitudinal data. Extensive data examples and simulations support the effectiveness of the approach proposed.
Confidence Bands in Non-Parametric Errors-In-Variables Regression
Authors: Delaigle Aurore , Hall Peter , Jamshidi Farshid
Published: April 2014

Errors-in-variables regression is important in many areas of science and social science, e.g. in economics where it is often a feature of hedonic models, in environmental science where air quality indices are measured with error, in biology where the vegetative mass of plants is frequently obscured by mismeasurement and in nutrition where reported fat intake is typically subject to substantial error. To date, in non-parametric contexts, the great majority of work has focused on methods for estimating the mean as a function, with relatively little attention being paid to techniques for empirical assessment of the accuracy of the estimator. We develop methodologies for constructing confidence bands. Our contributions include techniques for tuning parameter choice aimed at minimizing the coverage error of confidence bands.
Bootstrapping Locally Stationary Processes
Authors: Kreiss Jens-Peter , Paparoditis Efstathios
Published: April 2014

We propose a non-parametric method to bootstrap locally stationary processes which combines a time domain wild bootstrap approach with a non-parametric frequency domain approach. The method generates pseudotime series which mimic (asymptotically) correct, the local second- and to the necessary extent the fourth-order moment structure of the underlying process. Thus it can be applied to approximate the distribution of several statistics that are based on observations of the locally stationary process. We prove a bootstrap central limit theorem for a general class of statistics that can be expressed as functionals of the preperiodogram, the latter being a useful tool for inferring properties of locally stationary processes. Some simulations and a real data example shed light on the finite sample properties and illustrate the ability of the bootstrap method proposed.
Distinctive erosional and depositional structures formed at a canyon mouth: A lower Pleistocene deep‐water succession in the Kazusa forearc basin on the Boso Peninsula, Japan
Authors: Ito Makoto , Ishikawa Kazuaki , Nishida Naohisa
Published: April 2014

The canyon mouth is an important component of submarine‐fan systems and is thought to play a significant role in the transformation of turbidity currents. However, the depositional and erosional structures that characterize canyon mouths have received less attention than other components of submarine‐fan systems. This study investigates the facies organization and geometry of turbidites that are interpreted to have developed at a canyon mouth in the early Pleistocene Kazusa forearc basin on the Boso Peninsula, Japan. The canyon‐mouth deposits have the following distinctive features: (i) The turbidite succession is thinner than both the canyon‐fill and submarine‐fan successions and is represented by amalgamation of sandstones and pebbly sandstones as a result of bypassing of turbidity currents. (ii) Sandstone beds and bedsets show an overall lenticular geometry and are commonly overlain by mud drapes, which are massive and contain fewer bioturbation structures than do the hemipelagic muddy deposits. (iii) The mud drapes have a microstructure characterized by aggregates of clay particles, which show features similar to those of fluid‐mud deposits, and are interpreted to represent deposition from fluid mud developed from turbidity current clouds. (iv) Large‐scale erosional surfaces are infilled with thick‐bedded to very thick‐bedded turbidites, which show lithofacies quite similar to those of the surrounding deposits, and are considered to be equivalent to scours. (v) Concave‐up erosional surfaces, some of which face in the upslope direction, are overlain by backset bedding, which is associated with many mud clasts. (vi) Tractional structures, some of which are equivalent to coarse‐grained sediment waves, were also developed, and were overlain locally by mud drapes, in association with mud drape‐filled scours, cut and fill structures and backset bedding. The combination of these outcrop‐scale erosional and depositional structures, together with the microstructure of the mud drapes, can be used to identify canyon‐mouth deposits in ancient deep‐water successions.
Influence of the HLA characteristics of Italian patients on donor search outcome in unrelated hematopoietic stem cell transplantation
Authors: Testi M. , Andreani M. , Locatelli F. , Arcese W. , Troiano M. , Battarra M. , Gaziev J. , Lucarelli G.
Published: April 2014

The information regarding the probability of finding a matched unrelated donor (MUD) within a relatively short time is crucial for the success of hematopoietic stem cell transplantation (HSCT), particularly in patients with malignancies. In this study, we retrospectively analyzed 315 Italian patients who started a search for a MUD, in order to assess the distribution of human leukocyte antigen (HLA) alleles and haplotypes in this population of patients and to evaluate the probability of finding a donor. Comparing two groups of patients based on whether or not a 10/10 HLA‐matched donor was available, we found that patients who had a fully‐matched MUD possessed at least one frequent haplotype more often than the others (45.6% vs 14.3%; P = 0.000003). In addition, analysis of data pertaining to the HLA class I alleles distribution showed that, in the first group of patients, less common alleles were under‐represented (20.2% vs 40.0%; P = 0.006). Therefore, the presence of less frequent alleles represents a negative factor for the search for a potential compatible donor being successful, whereas the presence of one frequent haplotype represents a positive predictive factor. Antigenic differences between patient and donor observed at C and DQB1 loci, were mostly represented by particular B/C or DRB1/DQB1 allelic associations. Thus, having a particular B or DRB1 allele, linked to multiple C or DQB1 alleles, respectively, might be considered to be associated with a lower probability of a successful search. Taken together, these data may help determine in advance the probability of finding a suitable unrelated donor for an Italian patient.
The Bothnian coupled oroclines of the Svecofennian Orogen: a Palaeoproterozoic terrane wreck
Authors: Lahtinen Raimo , Johnston Stephen T. , Nironen Mikko
Published: April 2014

The accretion of magmatic arcs gives rise to elongate, linear orogens and is a key process in forming new continental crust. Many Precambrian continents are, however, presently equidimensional or have large areas without any clear linearity, such as the central part of the Palaeoproterozoic Svecofennian Orogen (1.92–1.77 Ga). One way of forming an equidimensional continental domain is by buckling of a linear orogen about vertical axes of rotation into one or more coupled oroclines. Here, we reinterpret existing data and demonstrate the occurrence of coupled Bothnian oroclines in the Svecofennian Orogen. Palinspastic restoration of the southern and northern Bothnian oroclines brings a 1000‐km‐long segment of the Svecofennian Orogen into an originally linear, NW‐striking geometry that restores the lithological belts, metamorphic zones and structural vergences to a common direction, and which indicates that the orogen consists of a SW‐facing arc, which has been shortened along NE‐verging folds and thrust faults.
Spatio‐Temporal Building Population Estimation for Highly Urbanized Areas Using GIS
Authors: Greger Konstantin
Published: April 2014

Detailed population information is crucial for the micro‐scale modeling and analysis of human behavior in urban areas. Since it is not available on the basis of individual persons, it has become necessary to derive data from aggregated census data. A variety of approaches have been published in the past, yet they are not entirely suitable for use in the micro‐scale context of highly urbanized areas, due mainly to their broad spatial scale and missing temporal scale. Here we introduce an enhanced approach for the spatio‐temporal estimation of building populations in highly urbanized areas. It builds upon other estimation methodologies, but extends them by introducing multiple usage categories and the temporal dimension. This allows for a more realistic representation of human activities in highly urbanized areas and the fact that populations change over time as a result of these activities. The model makes use of a variety of micro‐scale data sets to operationalize the activities and their spatio‐temporal representations. The outcome of the model provides estimated population figures for all buildings at each time step and thereby reveals spatio‐temporal behavior patterns. It can be used in a variety of applications concerning the implications of human behavior in urban areas.
Measuring the Reliability of Wheelchair User Route Planning based on Volunteered Geographic Information
Authors: Neis Pascal
Published: April 2014

The development of a wheelchair user friendly route planning application inherits a number of special requirements and details that need to be considered during the generation of the routing graph and the corresponding algorithm, making this task much more complex than car or pedestrian related applications. Each wheelchair type and, more importantly, each individual user might have different needs with regards to the way condition or other criteria. This study proposes a new approach to route planning for wheelchair users tailored for individual and personal requirements provided by the user and the calculation of a reliability factor of the computed wheelchair path. The routing graph is based on the freely available Volunteered Geographic Information (VGI) retrieved from the OpenStreetMap (OSM) project. The newly created algorithm is evaluated and tested for a selected area in Bonn, Germany. A new reliability factor is introduced that gives direct feedback about the quality of the generated path. Similar factor estimations can also be utilized for multiple route planning applications where VGI or other commercial or administrative data is implemented and more detailed factors than a simple geometric representation of a street network are of importance.
Going for gold: blood planning for the London 2012 Olympic Games
Authors: Glasgow S. M. , Allard S. , Rackham R. , Doughty H.
Published: April 2014

The Olympics is one of the largest sporting events in the world. Major events may be complicated by disruption of normal activity and major incidents. Health care and transfusion planners should be prepared for both. Previously, transfusion contingency planning has focused on seasonal blood shortages and pandemic influenzas. This article is the first published account of transfusion contingency planning for a major event. We describe the issues encountered and the lessons identified during transfusion planning for the London 2012 Olympics.

Planning was started 18 months in advance and was led by a project team reporting to the Executive. Planning was based on three periods of Gamestime. The requirements were planned with key stakeholders using normal processes enhanced by service developments. Demand planning was based on literature review together with computer modelling. The aim was blood‐stock sufficiency complimented by a high readiness donor panel to minimise waste. Plans were widely communicated and table‐top exercised.

Full transfusion services were maintained during both Games with all demands met. The new service improvements and high readiness donors worked well. Emergency command and control have been upgraded. Red cell concentrate (RCC) stock aged but wastage was not significantly increased. The key to success was: early planning, stakeholder engagement, service developments, integration of transfusion service planning within the wider health care community and conduct within an assurance framework.
Immune trypanolysis test with blood spotted on filter paper for epidemiological surveillance of sleeping sickness
Authors: Camara Oumou , Camara Mamadou , Lejon Veerle , Ilboudo Hamidou , Sakande Hassane , Léno Mamadou , Büscher Philippe , Bucheton Bruno , Jamonneau Vincent
Published: April 2014

Objectives

The immune trypanolysis test (TL) is an accurate sero‐diagnostic tool increasingly implemented for sleeping sickness medical surveillance, but it is restricted to the reference laboratories. To facilitate storage and transport of the test specimen, we developed a protocol for the examination of blood spotted on filter paper (TL‐fp) that can be stored and shipped at ambient temperature. We compared its performance with the classical TL on plasma (TL‐pl) that needs to be kept frozen until use.

Methods

The study was conducted in active foci of the Republic of Guinea. In total, 438 specimens from treated and untreated sleeping sickness patients and serological suspects were tested with both methods.

Result

TL‐fp gave significantly less positive results than TL‐pl, but all the confirmed sleeping sickness cases were positive with the TL‐fp protocol.

Conclusion

TL‐fp appears to offer a good compromise between feasibility and sensitivity to detect currently infected subjects who play a role in the transmission of Trypanosoma brucei gambiense and is useful for contributing to the elimination of gambiense sleeping sickness.
Policy challenges facing integrated community case management in Sub‐Saharan Africa
Authors: Bennett Sara , George Asha , Rodriguez Daniela , Shearer Jessica , Diallo Brahima , Konate Mamadou , Dalglish Sarah , Juma Pamela , Namakhoma Ireen , Banda Hastings , Chilundo Baltazar , Mariano Alda , Cliff Julie
Published: April 2014

Objective

To report an in‐depth analysis of policy change for integrated community case management of childhood illness (iCCM) in six sub‐Saharan African countries. We analysed how iCCM policies developed and the barriers and facilitators to policy change.

Methods

Qualitative retrospective case studies drawing from document reviews, semi‐structured interviews and in‐country validation workshops were conducted in Burkina Faso, Kenya, Malawi, Mali, Mozambique and Niger. These countries were selected to maximise variation in iCCM policy status, community health worker (CHW) models and different African regions.

Results

Country iCCM policies evolved in an ad hoc fashion, but were substantially influenced by the history of primary health care and the nature of CHW programmes. Technical officers within Ministries of Health led iCCM policy change with support from international donors, but neither communities nor political leadership was mobilised. Concerns about achieving the Millennium Development Goals, together with recognition of the shortcomings of existing child health programmes, led to the adoption of iCCM policies. Availability of external financing played a critical role in facilitating policy change.

Conclusions

iCCM policy change has been promoted by international agencies, but national governments have struggled to align iCCM with country health systems. Greater investment is needed in tailoring global policy initiatives to match country needs. High‐level, political ownership of iCCM policies could facilitate policy change, as could clearer strategies for ensuring the long‐term sustainability of such policies.
Epigenome‐wide DNA methylation assay reveals placental epigenetic markers for noninvasive fetal single‐nucleotide polymorphism genotyping in maternal plasma
Authors: Ou Xueling , Wang Huan , Qu Dongyang , Chen Yongzhen , Gao Jun , Sun Hongyu
Published: April 2014

Background

The use of DNA methylation difference between maternal blood cell and fetal (placental) DNA is one of the main areas of interest for the development of fetal epigenetics markers in maternal plasma.

Study Design and Methods

We employed a methylation array (HumanMethylation450 array, Illumina, Inc.) to identify novel biomarkers that are specially hypermethylated in placental DNA versus maternal blood cells in a genome‐wide basis. Validation by bisulfite genomic sequencing was performed and the priority was given to potential targets that harbor differential methylated CpG sites overlapped with at least two methylation‐sensitive restriction enzyme (MSRE) recognizing sites, as well as one polymorphic single‐nucleotide polymorphism (SNP), within a short DNA stretch. Three candidate regions of PSMB8, SKI, and CHST11 gene were selected for developing a preliminary polymerase chain reaction assay with MSRE digestion of maternal plasma DNA. SNP genotypes were confirmed by direct sequencing.

Results

We identified 2944 and 5218 fetal‐specific hypermethylated CpG sites in the first‐ and third‐trimester placenta, respectively, of which 2613 were overlapped, suggesting a consistency of differential methylation during the whole pregnancy. The array results were confirmed by bisulfite genomic sequencing. The preliminary tests in maternal plasma showed that postdigestion hypermathylated versions of these candidate molecules were detectable only in pregnant women. We further revealed that methylated targets in maternal plasma possessed the fetal SNP genotypes.

Conclusion

The present studies systematically identified hypermethylated sites in fetal tissues and preliminarily demonstrated that some of the fetal epigenetic markers that contain informative SNPs have great potential for noninvasive fetal genetic diagnosis.
A case of high‐titer anti‐D hemolytic disease of the newborn in which late onset and mild course is associated with the D variant, RHD‐CE(9)‐D
Authors: Jakobsen Marianne A. , Nielsen Christian , Sprogøe Ulrik
Published: April 2014

Background

The RhD antigen is very immunogenic and is a significant cause of hemolytic disease of the newborn (HDN). The RHD‐CE(8‐9)‐D hybrid allele is commonly associated with a D– phenotype. Here, we report a case of high‐titer maternal anti‐D and late onset of HDN in a newborn carrying a RHD‐CE(9)‐D variant supposedly encoding the same partial D antigen as the RHD‐CE(8‐9)‐D allele, but with significant expression of D antigen.

Study Design and Methods

To elucidate the blood group antigen background of the case, we carried out serologic, flow cytometric, and genetics studies of the newborn and his father.

Conclusion

Individuals carrying the RHD‐CE(9)‐D allele do express D antigen, but do so at significantly lower levels than those carrying the more common D+ phenotypes (e.g., DCe/dce). It may mitigate and delay otherwise severe HDN in pregnancies complicated by high‐titer anti‐D.
Topiramate in the management of feline idiopathic ulcerative dermatitis in a two‐year‐old cat
Authors: Grant David , Rusbridge Clare
Published: April 2014

Background

Feline idiopathic ulcerative dermatosis is a rare, poorly understood condition characterized by self‐trauma. The lesion presents as a nonhealing, crusted ulcer, which occurs most commonly on the dorsal midline of the neck or between the scapulae.

Animal

A 2‐year‐old female neutered domestic short hair cat was presented with an ulcerative dermatosis affecting the dorsal midline. Previous investigations had failed to identify the cause, and the lesion was resistant to treatment.

Methods and results

Diagnosis was based on clinical findings and confirmed by histopathology showing epidermal ulceration and superficial necrosis with a mild dermal infiltrate together with subepidermal fibrosis. The cat had been fed a commercial hypoallergenic diet for 6 months, which had successfully managed its chronic diarrhoea. Deep skin scrapings, cytology and fungal culture failed to demonstrate pathogens.

Conclusions and clinical importance

Remission was obtained within 4 weeks and has been maintained over a 30 month period with topiramate (5 mg/kg orally twice daily), an anti‐epileptic drug used in human medicine. Attempts to withdraw this therapy led to relapse within 24 h on two occasions. To the best of the authors' knowledge, this is the first case report of the use of this drug for feline idiopathic ulcerative dermatosis.
Pulsed ultraviolet light inactivation of Pseudomonas aeruginosa and Staphylococcus aureus biofilms
Authors: Garvey Mary , Rabbitt Danny , Stocca Alessia , Rowan Neil
Published: April 2014

Microbial biofilms are complex communities that form when planktonic bacterial species attach to surfaces in many settings where they can provide a source of pathogenicity. The relative ineffectiveness of conventional disinfectants such as free chlorine and monochloramine for the inactivation of some species found in water has led to evaluation of alternative disinfectants for drinking and wastewater treatment. In recent years, novel pulsed power electrotechnologies have been introduced and are being considered as possible alternatives to current methods for inactivating problematic species in water. This study focuses on the ultraviolet (UV) inactivation of bacterial biofilms using a pulsed UV light approach as a potential disinfection method for water treatment operating systems. Biofilms were stimulated to form attached to polyvinyl chloride coupons using a recommended Centre for Disease Control biofilm reactor followed by exposure to a range of UV doses. Findings show that this method is highly effective at inactivating both planktonic and biofilm cells with significant inactivation rates obtained for both test species. Specifically, a 7.2 and 5.9 log10 inactivation was achieved with up to 21.6 μJ/cm2 UV for Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Findings from this study highlight the effectiveness of pulsed UV for the inactivation of Pseudomonas biofilms among other test species. Research conducted by this group suggests that this pulsed UV system may offer a useful method for the disinfection of drinking and wastewater supplies.
Evaluating the functionality of xeno‐relevant transgenes
Authors: Baehr Andrea , Wuensch Annegret , Kemter Elisabeth , Kessler Barbara , Kurome Mayuko , Seissler Jochen , Ayares David , Wolf Eckhard , Klymiuk Nikolai
Published: April 2014

With the scientific progress made in recent years, clinical application of xenotransplantation has become a realistic goal. Besides optimizing immunosuppressive regimens, the development of geneticallytailored donor pigs contributed to this success. Within the CRC 127 “Biology of xenogeneic cell, tissue and organ transplantation – from bench to bedside” we aim at developing novel transgenic donor pigs for islet transplantation, and providing existing transgenic donor lines to German as well as international collaboration partners.

Regarding novel donor models, we have established transgenic pigs carrying the x‐linked inhibitor of apoptosis (XIAP) under the control of the porcine insulin promoter to achieve beta‐cell specific expression, as apoptosis has been postulated to play a major role in islet loss during isolation, in vitro cultivation and engraftment. Expression analyses of the first transgenic founders are presently underway. In addition, an advanced donor pig aiming at the temporary expression of vascular endothelial growth factor (VEGF) for improved engraftment of islets is being established. The model is based on the TetOn principle which requires two independent transgenes. First litters of founder pigs carrying a construct for beta‐cell specific expression of the transactivator (TA) have been delivered and qPCR analysis of the pancreas revealed different levels of TA expression in individual animals. Primary cells from a highly expressing founder will be transfected with a construct carrying the regulated VEGF gene under the control of the TA response element.

For detailed pre‐clinical evaluation of already characterized transgenic pigs, we have established multitransgenic lines in collaboration with Revivicor Inc. carrying either aGalTKO/CD46/hTM, aGalTKO/CD46/INS‐LEA or aGalTKO/CD46/HLA‐E. We provide these animals, in addition to single‐transgenic INS‐LEA pigs, to the groups of P.Brenner and J.Seissler within the consortium. Furthermore, multi‐transgenic animals are provided to the groups of M.Mohiuddin, D.K.C. Cooper and R. Pierson by D. Ayares and islets of INS‐LEA expressing pigs are supplied to B. Hering in collaboration with J. Seissler.

The ongoing evaluation of existing transgenes or their combination will reveal more detailed insight into the complexity of rejection mechanisms and, thus, support the design of optimized future transgenic approaches.
Bio artificial pancreas – the immune barrier and oxygen deficiency
Authors: Barkai Uriel , Ludwig Barbara , Bornstein Stefan R. , Zimermann Baruch , Gendler Zohar , Rotem Avi
Published: April 2014

Background

Xenogeneic transplantation of islets of Langerhans into type I diabetes patients imposes a major challenge. This is even more exaggerated when the target is set as xenotransplantation of islets in relatively oxygen poor environment without the use of immunosuppressive therapy. To overcome these hurdles, adequate encapsulation technique and active oxygen supply should be employed.

Methods

A two‐article device was developed (the βAir). The first contains an implantable bioreactor module comprising of islets of Langerhans encapsulated in alginate hydrogel and protected from the host immune system by a complex membrane. The second is an external oxygen injection device for replenishing of the islet biomass in the implantable device once a day.

Results

Both small and large diabetic animal models (rats and pigs) were experimented. The βAir device proved: (i) ability to maintain near normal glycemic control in rats transplanted with either isogeneic or allogeneic islets for a period of over 6 months and in pigs transplanted with rat islets using marginal dose of islets (~6,500 IEQ/kg) for a period of up to 75 days; (ii) ability to maintain adequate oxygen tension required to nourish the islets graft for a period of 24 h while maintaining optimal islet's function. (iii) long term separation of transplanted graft from host immune system thus maintaining normal islets function in rat allotype, in rat‐to‐pig and in human‐to‐rat transplantation systems and (iv) high level of biocompatibility and tolerability of the device.

Conclusion

The βAir device proved to offer functional solution to islets transplantation in allogeneic and xenogeneic setting. It may provide a solution to transplantation of porcine islets to diabetic patients.

References

[1] Barkai U, Weir GC, Colton CK et al. Enhanced oxygen supply improves islet viability in a new bioartificial pancreas. Cell Transplant. 2013; 22: 1463–1476.

[2] Neufeld T, Ludwig B, Barkai U et al. The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs. PLoS One. 2013; 8(8): e70150.
Reduction of xeno‐antigens in porcine pulmonary heart valves by decellularization and glycolytic enzymatic treatment
Authors: Hilfiker Andres , Ramm Robert , Findeisen Katja , Goecke Tobias , Haverich Axel
Published: April 2014

Background

Heart valve replacement therapy with allogenic decellularized heart valve matrices allows an in vivo autologisation by repopulation of the foreign matrix with autologous cells. This method, which is superior based on experimental evidence to traditional heart valve replacements either of mechanical or glutaraldehyde fixated heart valve grafts, is current object of a multicentric international clinical trial (ESPOIR) granted by the EU. To circumvent the limited availability of allogenic heart valve matrices, we aim to the reduction of immunogenic epitops common to xenogeneic materials by high efficient decellularization paralleled by enzymatic treatment of the glycocalyx.

Methods

Porcine pulmonary heart valves of landrace pigs harvested at the local slaughterhouse were subjected to detergent and proteinase based decellularization protocols widely described in the literature. In a second step decellularized matrices were exposed to α1‐3 Galactosidase or PNGase F digestion. Alterations in αGal epitope levels and glycocalyx structures were investigated by inhibitory ELISAs with anti‐αGal antibodies (M86) and IgGs of unconditioned human sera on crushed matrix, and histochemical stains on cryosections exploiting lectins as isolectin B4 (staining αGal), wheat germ agglutinin (staining GalNAc), and Datura stramonium lectin (staining GlcNAc). Equally treated GalT‐KO pig and human pulmonary artery tissues served as controls.

Results

Inhibitory ELISA results show that all decellularization protocols reduced αGal epitope levels significantly, some down to <30% of native porcine tissue. Further reduction down to control levels was achieved by α1‐3 Galactosidase digestion. The absence of preformed non‐ αGal xenoantibodies in human IgG of unconditioned donors is evident by equal binding of human IgG to decellularized tissues of GalT‐KO and human origin.

Histochemistry reveals that decellularization results in reduced Isolectin B4 (αGal) and WGA (GalNAc) staining, but not of DSL (GlcNAc) staining. Additional enzymatic treatment of decellularized matrix with PNGase F reduces binding of all lectins, whereas digestion with α1‐3 Galactosidase only affects Isolectin B4 staining (αGal).

Conclusion

The reduction of αGal epitope levels is strongly dependent on the decellularization method. A further decrease can be achieved by enzymatic digestion of carbohydrates present on the decellularized matrix. Sugar structures of the glycocalyx are believed to be strongly immunogenic, therefore, their general removal by PNGase F digestion may produce non‐immunogenic heart valve matrices. However, the lack of preformed human non‐ αGal xenoantibodies in non conditioned sera requires the testing for immunogenicity in an in vivo system. Thus, in future decellularized and enzymatically treated GalT‐KO matrices will be tested in a humanized mouse model.
Ex‐vivo testing and preclinical heterotopic thoracic cardiac xenotransplantation of multi‐transgenic organs
Authors: Abicht Jan‐Michael , Mayr Tanja , Guethoff Sonja , Buchholz Stefan , Werner Fabian , Baehr Andrea , Klymiuk Nikolai , Wuensch Annegret , Wolf Eckhart , Belka Claus , Ayares David , Petersen Björn , Niemann Heiner , Bongoni Anjan K. , Rieben Robert , McGregor Christopher , Reichart Bruno , Brenner Paolo
Published: April 2014

The heterotopic thoracic pig‐to‐baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi‐transgenic porcine donor hearts in an ex‐vivo perfusion model first.

Ex‐vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package.

Pig‐to‐baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal‐KO/hCD46), and two triple (Gal‐KO/hCD46/hTM resp./HLA‐E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti‐CD20‐antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti‐CD20‐antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five.

The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies.
B cell activation and induced antibody responses to porcine antigen can be diminished by PD‐L1‐mediated triggering of PD‐1
Authors: Buermann Anna , Borns Katja , Römermann Dorothee , Plege‐Fleck Annegret , Schwinzer Reinhard
Published: April 2014

Background

T cell activation is regulated by the integration of positive and negative signals. A strategy to down‐regulate human immune responses to a porcine xenograft is the enhancement of negative costimulatory signals. The programmed death‐1 receptor (PD‐1) is one of the known negative regulators for T cell activation and we have previously shown that human in vitro T cell responses to porcine transfectants over‐expressing human PD‐Ligand 1 (PD‐L1) are reduced as compared to responses triggered by control cells. However, PD‐1 is also expressed on B cells and its role in B cell activation is not well understood so far. Thus, the present study focused on the effects of PD‐1/PD‐L1 targeting on the humoral immune response to xenoantigens.

Methods

In vitro activation of human B cells was induced by co‐culturing with porcine L23 cells (B cell line) genetically engineered to over‐express human PD‐L1 (L23‐PD‐L1 cells) or “mock” ‐transfected controls. In vivo immune responses to the two cell types were characterized in a rat model by immunization experiments. The anti‐pig antibody titer in the culture supernatant or the sera of rats was examined by binding to L23 cells and visualized by flow cytometry using FITC‐conjugated IgM and IgG.

Results

Stimulation of human peripheral blood mononuclear cells (hPBMC) with L23 cells resulted in up‐regulation of the early activation marker CD69 on CD19+ B cells. Furthermore, we observed T cell dependent proliferation of B cells and differentiation to antibody secreting cells in vitro. This was shown by the detection of anti‐pig antibodies in the culture supernatant of hPBMCs stimulated with xenoantigen after 7 days and the titer increased till day 12, with higher amounts of IgM than IgG antibodies. When the activation of B cells was induced by L23‐PD‐L1 cells, lower antibody titers were determined in comparison to B cell activation induced by L23 control cells. Moreover, the humoral immune response was reduced when a soluble PD‐L1 molecule was applied to hPBMCs stimulated with porcine cells. To further characterize the antibody responses to L23‐PD‐L1 and control cells in vivo, we analyzed the level of anti‐pig antibodies in rats after cell transplantation under the kidney capsule. In line with the in vitro data we found lower levels of antibodies in the serum of rats immunized with L23‐PD‐L1 cells than in rats receiving L23 control cells.

Conclusion

These data suggest that the antibody response to porcine xenoantigen is diminished in the presence of the inhibitory ligand PD‐L1. Application of PD‐L1 might be a strategy to prevent induction of anti‐graft antibody responses, which are particularly strong in the pig‐to‐primate combination and represent a significant immunologic barrier for clinical xenotransplantation.
Decellularization followed by PNGase F treatment efficiently removes immunogenic αGal epitopes and other N‐acetylglucosamine structures on the glycocalyx of porcine pulmonary heart valve matrices
Authors: Findeisen Katja , Ramm Robert , Hilfiker Andres
Published: April 2014

Background

Xenotransplantation leads to hyperacute or acute graft reaction due to interaction of preformed human antibodies, mainly αGal‐antibodies, with carbohydrate structures present on non‐human tissue. By removing these carbohydrate structures from porcine decellularized pulmonary heart valves (PHV), grafts might be generated that allow heart valve replacement therapy as with allogeneic PHV matrices.

Methods

Thus, after detergents based decellularization (0.5%SDS/0.5%Triton‐X100) cell free PHV matrices were enzymatically treated with α1‐3,6‐galactosidase or PNGase F. The potential impact on the glycocalyx was investigated by histochemical stains utilizing isolectin B4 (IL‐B4), wheat germ agglutinin (WGA), Datura stramonium lectin (DSL), and Ricinus communis agglutinin (RCA I) on decellularized only, and decellularized and enzymatically treated specimens. Native PHV tissue served as controls.

Results

All used lectins stained native heart valve tissue. Decellularization resulted in reduced IL‐B4 and WGA staining, whereas cell removal had no effect on DSL and RCA I staining. Enzymatic PNGase F treatment resulted in a further decrease of IL‐B4 and WGA staining whereas initially not affected DSL stain was reduced as well. Enzymatic treatment with α1‐3,6‐galactosidase led to a reduction of IL‐B4 stain only.

Conclusion

Decellularization per se is able to reduce αGal epitopes as demonstrated by IL‐B4 staining. This reduction of αGal epitopes can be enhanced by α1‐3,6‐galactosidase digestion as expected. Enzymatic treatment with PNGase F that recognizes GlcNacβ(1‐N)Asn sites results in removal of carbohydrate structures as αGal and N‐acetyl‐glucosamines to a high degree as demonstrated by IL‐B4, WGA, and DSL stains.

In summary, detergent based decellularization followed by PNGase F treatment resembles an efficient way to remove immunogenic epitopes from porcine pulmonary heart valve matrices, thus potentially enabling the generation of xenogeneic PHV matrices for clinical application.