How to Perform Umbilical Cord Arterial and Venous Blood Sampling in Neonatal Foals

Umbilical cord arterial and venous blood gas analysis is a commonly performed procedure in human neonatal medicine to help ascertain a newborn infant’s oxygenation and acid-base status prior to birth. Defined protocols for performing the procedure have been described in the medical literature. The aim of this report was to describe in detail the procedure for collecting paired blood samples from the umbilical artery and vein in newborn foals so that stall-side blood gas analysis could be carried out. Thirty-five Thoroughbred foals >320 days gestation from mares at one stud farm were sampled. Paired umbilical arterial and venous whole-blood samples were obtained in n=30 foals, umbilical artery only samples obtained in n=3 and umbilical vein only samples obtained in n=2 foals. There were no adverse events or clinical outcomes associated with the sampling protocol described. The authors found that umbilical cord blood collection for blood gas analysis was a practical clinical technique that potentially could be used as a stall-side method for assessing the in utero oxygenation and acid-base status of newborn foals

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry

Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at ?15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at ∼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules

Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire

Idiosyncratic adverse drug reactions are unpredictable, dose independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkage between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells that required HLA-B*57:01 molecules for their function. However, the mechanism by which abacavir induces this pathologic T cell response remains unclear. Here we show that abacavir can bind within the F-pocket of the peptide-binding groove of HLA-B*57:01 thereby altering its specificity. This supports a novel explanation for HLA-linked idiosyncratic adverse drug reactions; namely that drugs can alter the repertoire of self-peptides presented to T cells thus causing the equivalent of an alloreactive T cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir, and that were recognized by T cells of hypersensitive patients. The assays we have established can be applied to test additional compounds with suspected HLA linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA linked hypersensitivities as well as guide the development of safer drugs

Geometrical exponents of contour loops on synthetic multifractal rough surfaces: multiplicative hierarchical cascade p-model

In this paper, we study many geometrical properties of contour loops to characterize the morphology of synthetic multifractal rough surfaces, which are generated by multiplicative hierarchical cascading processes. To this end, two different classes of multifractal rough surfaces are numerically simulated. As the first group, singular measure multifractal rough surfaces are generated by using the $p$ model. The smoothened multifractal rough surface then is simulated by convolving the first group with a so-called Hurst exponent, $H^*$ . The generalized multifractal dimension of isoheight lines (contours), $D(q)$, correlation exponent of contours, $x_l$, cumulative distributions of areas, $\xi$, and perimeters, $\eta$, are calculated for both synthetic multifractal rough surfaces. Our results show that for both mentioned classes, hyperscaling relations for contour loops are the same as that of monofractal systems. In contrast to singular measure multifractal rough surfaces, $H^*$ plays a leading role in smoothened multifractal rough surfaces. All computed geometrical exponents for the first class depend not only on its Hurst exponent but also on the set of $p$ values. But in spite of multifractal nature of smoothened surfaces (second class), the corresponding geometrical exponents are controlled by $H^*$, the same as what happens for monofractal rough surfaces.Comment: 14 pages, 14 figures and 6 tables; V2: Added comments, references, table and major correction

In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Laminitis in dairy goats (Capra aegagrus hircus) on a low-forage diet

Dairy goats on high-concentrate diets attain high production levels, but at what cost? Here, ongoing lameness problems in a herd offered ad lib concentrates and roughages throughout their lifetime were investigated. Five severely affected, chronically lame animals were euthanased and examined postmortem. Foot pathology consisted of distortion of the claw shape and irregular fissures over the solar and bulbar horn with the distal phalanx rotated downwards on two claws. Rumen pH was measured between 5.26 and 5.46 with moderate rumen mucosa hyperkeratosis, and ulcerative, mild lymphocytic rumenitis. Feet showed irregular hyperplasia of the epidermal laminae with parakeratotic hyperkeratosis, especially in solar regions. Dense clusters of lymphocytes expanded the dermal laminae. Based on these findings, chronic laminitis was suspected. Ruminal hyperkeratosis was likely a result of prolonged periods of acidosis. The consequences of feeding a high-concentrate ration throughout the life of dairy goats need more research

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response In Vitro and In Vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-γ production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c+ DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 µm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-γ production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response