Low levels of naturally occurring regulatory T lymphocytes in blood of mares with early pregnancy loss.

Early pregnancy loss is a major reason for low reproductive efficiency in the horse. In humans and mice, low numbers of regulatory T cells (Treg cells) are linked to miscarriage. The percentage of Treg cells in oestrous mares at the start of the breeding season was evaluated in relation to the outcome of subsequent pregnancy. For identification and quantification of Treg cells, a highly sensitive and specific qPCR assay targeting the Treg-specific demethylated region in the equine forkhead box transcription factor (FOXP3) gene was established. In a total of 108 mares, pregnancy was followed until detection of early pregnancy loss (n=17), abortion without identification of an infectious or apparent cause (n=9) or birth of a viable foal (n=82). Measured Treg-cell levels did not significantly differ between mares that conceived (82%; 1.50±0.04%) or did not get pregnant (18%; 1.45±0.10%). The Treg-cell percentage at oestrus before breeding was significantly different (P<0.05) between mares that either underwent early pregnancy loss up to Day 40 of pregnancy (1.29±0.07%) and mares that aborted (1.61±0.15%) or gave birth to a live foal (1.52±0.05%). These results suggest that low levels of Treg cells in mares can contribute to pregnancy loss up to Day 40 after ovulation.

Case Report: Primary Diffuse Leptomeningeal Oligodendrogliomatosis in a Young Adult Cat.

A 2-year-old cat was presented with progressive ataxia. Despite treatment the animal died. Pathomorphological examination revealed a widespread leptomeningeal mass at all levels of the central nervous system accentuated on the cervical spinal cord and the medulla oblongata without presence of a primary intraaxial tumor. The neoplasm was mainly composed of round, uninucleate cells with hyperchromatic nuclei, which were immunopositive for OLIG2, doublecortin, MAP2, synaptophysin, and vimentin, indicating components of both oligodendroglial and neuronal differentiation. Ki-67 immunohistochemistry indicated a high proliferation activity of the neoplasm. Few GFAP positive and Iba-1 positive cells were interpreted as reactive astrocytes and macrophages or microglia, respectively. The tumor was immunonegative for CD3, CD20, PAX5, MUM1, pan-cytokeratin, S100, NSE, p75NTR, NeuN and periaxin. These findings led to the diagnosis of primary diffuse leptomeningeal oligodendrogliomatosis. This is the first reported case of this entity in a young cat, which should be considered as a differential diagnosis for diffuse subarachnoidal round cell infiltrates.

T2 measurement in articular cartilage: impact of the fitting method on accuracy and precision at low SNR.

T2 relaxation time is a promising MRI parameter for the detection of cartilage degeneration in osteoarthritis. However, the accuracy and precision of the measured T2 may be substantially impaired by the low signal-to-noise ratio of images available from clinical examinations. The purpose of this work was to assess the accuracy and precision of the traditional fit methods (linear least-squares regression and nonlinear fit to an exponential) and two new noise-corrected fit methods: fit to a noise-corrected exponential and fit of the noise-corrected squared signal intensity to an exponential. Accuracy and precision have been analyzed in simulations, in phantom measurements, and in seven repetitive acquisitions of the patellar cartilage in six healthy volunteers. Traditional fit methods lead to a poor accuracy for low T2, with overestimations of the exact T2 up to 500%. The noise-corrected fit methods demonstrate a very good accuracy for all T2 values and signal-to-noise ratio. Even more, the fit to a noise-corrected exponential results in precisions comparable to the best achievable precisions (Cramér-Rao lower bound). For in vivo images, the traditional fit methods considerably overestimate T2 near the bone-cartilage interface. Therefore, using an adequate fit method may substantially improve the sensitivity of T2 to detect pathology in cartilage and change in T2 follow-up examinations.

Voxel-based reproducibility of T2 relaxation time in patellar cartilage at 1.5 T with a new validated 3D rigid registration algorithm.

OBJECT: T2 relaxation time is a promising MRI parameter for the early diagnosis and follow-up of osteoarthritis. Assessing the evolution of osteoarthritis needs exact comparison of datasets acquired at different times and knowledge of the T2 reproducibility. The aims of this work were to establish a method for voxel-wise comparison of T2 datasets and to assess voxel-based T2 reproducibility in healthy patellar cartilage. MATERIALS AND METHODS: A new rigid 3D-registration algorithm was developed. The precision of the registration algorithm was calculated with numerical simulations and in vitro measurements. In vivo T2 reproducibility was assessed in six volunteers measured at seven different times. The voxel-based reproducibility was characterized with the coefficient of variation (CV) of T2, and its regional variations were analyzed. RESULTS: The registration algorithm showed an average registration precision lower than 25% of the voxel size. In vivo voxel-based T2 reproducibility exhibited a median CV of 10.1%. Reproducibility showed significant regional differences. Largest CVs (15.4%) were found near the articular surface. The central regions showed the lowest CVs (7.2%) and the lateral regions intermediate CVs (11.2%). CONCLUSION: Using a rigid 3D-registration algorithm provides voxel-based T2 reproducibility errors comparable to former, 2D region-based approaches, thus opening the possibility of voxel-based monitoring of cartilage degradation in osteoarthritis.

An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose. METHODS: We introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control. RESULTS: Simultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical "chronotropic" compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient. DISCUSSION: Despite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential recording and a more reliable identification of drug effects on calcium handling.

T2 quantitation of human articular cartilage in a clinical setting at 1.5 T: implementation and testing of four multiecho pulse sequence designs for validity.

RATIONALE AND OBJECTIVES: Evaluation of the T2 relaxation time of articular cartilage holds great potential for quantitative assessment of internal changes of the cartilage matrix. The purpose of the present study was to assess the validity of multiecho-based cartilage T2 quantitation in a clinical MRI setting at 1.5 T. METHODS: Four multisection multiecho sequence variants dedicated for quantitative T2 mapping of human articular cartilage were implemented on a 1.5 T whole-body imager and tested for accuracy in CuSO4-agarose gel phantoms and human patellar cartilage. Sequence design was varied to minimize errors in T2 quantitation due to stimulated echoes. RESULTS: As compared with single spin-echo experiments, the apparent T2 values calculated from the multiecho sequence variants showed mean deviations ranging from +26% to -32% (phantoms) and from +42% to -18% (cartilage). The patellar cartilage T2 covered a range from about 25 milliseconds to 55 milliseconds, with longer T2 values observed in the more superficial layers. In cartilage, best results were obtained from the sequence design using improved section profiles and a spoiler gradient scheme for suppression of stimulated echoes. CONCLUSIONS: Our results revealed a clear dependence of apparent T2 relaxation times on the pulse sequence design, emphasizing that the "true" T2 is hard to find. In addition, the effect on the apparent T2 values resulting from the specific modification of any sequence variant varied according to the respective tissue's properties. Therefore, the acquisition technique in conjunction with the specific tissue on which T2 mapping is performed need to be reported in detail and should kept consistent to allow large-scale comparisons and monitoring of treatment strategies, e.g., in osteoarthritis.

Expanded clinical spectrum in hepatocyte nuclear factor 1b-maturity-onset diabetes of the young.

AIMS: HNF1B-maturity-onset diabetes of the young is caused by abnormalities in the HNF1B gene encoding the transcription factor HNF-1beta. We aimed to investigate detailed clinical features and the type of HNF1B gene anomaly in five pediatric cases with HNF1B-MODY. METHODS: From a cohort of 995 children and adolescents with diabetes, we analyzed the most frequent maturity-onset diabetes of the young genes (GCK, HNF1A, HNF4A) including HNF1B sequencing and deletion analysis by quantitative Multiplex-PCR of Short Fluorescent Fragments (QMPSF) if patients were islet autoantibody-negative and had one parent with diabetes or associated extrapancreatic features or detectable C-peptide outside honeymoon phase. Presence and size of disease-causing chromosomal rearrangements detected by QMPSF were further analyzed by array comparative genomic hybridization. RESULTS: Overall, five patients had a heterozygous HNF1B deletion, presenting renal disease, elevated liver enzymes, and diabetes. Diabetes was characterized by insulin resistance and adolescent onset of hyperglycemia. Additionally, clinical features in some patients were pancreas dysplasia and exocrine insufficiency (two of five patients), genital defects (three of five), mental retardation (two of five), and eye abnormalities (coloboma, cataract in two of five). One case also had severe growth deficit combined with congenital cholestasis, and another case had common variable immune deficiency. All patients reported here had monoallelic loss of the entire HNF1B gene. Whole genome array comparative genomic hybridization confirmed a precurrent genomic deletion of approximately 1.3-1.7 Mb in size. CONCLUSION: The clinical data of our cases enlarge the wide spectrum of patients with HNF1B anomaly. The underlying molecular defect in all cases was a 1.3- to 1.7-Mb deletion, and paired, segmental duplications along with breakpoints were most likely involved in this recurrent chromosomal microdeletion.

Focal epileptiform activity in the brain: detection with spike-related functional MR imaging--preliminary results.

The purpose of this study was to develop a spike-related functional magnetic resonance (MR) imaging method to detect epileptic brain activity. Correlations between simultaneous spike-related functional MR imaging and electroencephalographic (EEG) recordings were performed in 10 patients with focal epilepsy. Postprocessing techniques were implemented to eliminate contamination of the EEG recording from ballistocardiography and the echo-planar MR imaging sequence. A diagnostic EEG recording was achieved during functional MR imaging. Spike location correlated with the site of blood oxygen level-dependent signal increase. Spike-related functional MR imaging is a promising technique for detecting focal epileptic brain activity.

Mitochondrial DNA and the origins of the domestic horse.

The place and date of the domestication of the horse has long been a matter for debate among archaeologists. To determine whether horses were domesticated from one or several ancestral horse populations, we sequenced the mitochondrial D-loop for 318 horses from 25 oriental and European breeds, including American mustangs. Adding these sequences to previously published data, the total comes to 652, the largest currently available database. From these sequences, a phylogenetic network was constructed that showed that most of the 93 different mitochondrial (mt)DNA types grouped into 17 distinct phylogenetic clusters. Several of the clusters correspond to breeds and/or geographic areas, notably cluster A2, which is specific to Przewalski's horses, cluster C1, which is distinctive for northern European ponies, and cluster D1, which is well represented in Iberian and northwest African breeds. A consideration of the horse mtDNA mutation rate together with the archaeological timeframe for domestication requires at least 77 successfully breeding mares recruited from the wild. The extensive genetic diversity of these 77 ancestral mares leads us to conclude that several distinct horse populations were involved in the domestication of the horse.