Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay.

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Depletion of florfenicol in lactating dairy cows after intramammary and subcutaneous administration.

Eighteen Holstein dairy cows ranging in body weight from 500-700 kg and with an average milk yield of 37 ± 6 kg/day were used to investigate the depletion of florfenicol (FFL) in milk and plasma of dairy cows. Three groups of six were administered FFL: Group A, intramammary (IMM) infusion of ~2.5 mg FFL/kg BW at three consecutive milking intervals (total amount of ~7.5 mg/kg BW); Group B, one IMM infusion (20 mg/kg BW) into one quarter and Group C, one subcutaneous (SC) treatment (40 mg/kg BW). IMM infusions were into the right front quarter. Cows were milked daily at 06:00 and 18:00 h. The highest concentrations (Cmax ) and time to Cmax (Tmax ) were: 1.6 ± 2.2 µg·FFL/mL milk at 22 h (Group A), 5.5 ± 3.6 µg·FFL/mL milk at 12 h (Group B), and 1.7 ± 0.4 µg·FFL/mL milk at 12 h (Group C). The half-lives (t1/2 ) were ~19, 5.5, and 60 h, for Groups A, B, and C, respectively. FFL was below the limit of detection (LOD) by 60 h in three Group B cows, but above the LOD at 72, 84, and 120 h in three cows. FFL was above the LOD in milk from Group C's cows for 432-588 h. Plasma values followed the same trends as milk. The results demonstrate that IMM-infused FFL is bioavailable and below the LOD within 72-120 h. The concentration of FFL was detectable in both plasma and milk over the course of 2-3 weeks after SC administration. The absence of residue depletion data presents problems in determining safe levels of FFL residues in milk and edible tissues. The data presented here must not be construed as approval for extra-label use in food animals.

Overcoming the low relaxivity of gadofosveset at high field with spin locking.

The contrast agent gadofosveset, which binds reversibly to serum albumin, has a high longitudinal relaxivity at lower magnetic fields (≤3.0 T) but a much lower relaxivity at high fields. Spin locking is sensitive to macromolecular content; it is hypothesized that combining this technique with the albumin-binding properties of gadofosveset may enable increased relaxivity at high fields. In vitro measurements at 4.7 T found significantly higher spin-lock relaxation rates, R1ρ (1/T1ρ), when gadofosveset was serum albumin-bound than when unbound. R1ρ values for a nonbinding contrast agent (gadopentetate dimeglumine) in serum albumin were similar to those for unbound gadofosveset. R2 (1/T2) values were also significantly higher at 4.7 T for serum albumin-bound gadofosveset than for unbound. Spin locking at high field generates significantly higher relaxation rates for gadofosveset than conventional contrast agents and may provide a method for differentiating free and bound molecules at these field strengths.

Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.

The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E(2) (PGE(2)) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE(2) occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B(2) (TXB(2)). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE(2) in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFalpha) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE(2) production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.

A fast, model-independent method for cerebral cortical thickness estimation using MRI.

Several algorithms for measuring the cortical thickness in the human brain from MR image volumes have been described in the literature, the majority of which rely on fitting deformable models to the inner and outer cortical surfaces. However, the constraints applied during the model fitting process in order to enforce spherical topology and to fit the outer cortical surface in narrow sulci, where the cerebrospinal fluid (CSF) channel may be obscured by partial voluming, may introduce bias in some circumstances, and greatly increase the processor time required. In this paper we describe an alternative, voxel based technique that measures the cortical thickness using inversion recovery anatomical MR images. Grey matter, white matter and CSF are identified through segmentation, and edge detection is used to identify the boundaries between these tissues. The cortical thickness is then measured along the local 3D surface normal at every voxel on the inner cortical surface. The method was applied to 119 normal volunteers, and validated through extensive comparisons with published measurements of both cortical thickness and rate of thickness change with age. We conclude that the proposed technique is generally faster than deformable model-based alternatives, and free from the possibility of model bias, but suffers no reduction in accuracy. In particular, it will be applicable in data sets showing severe cortical atrophy, where thinning of the gyri leads to points of high curvature, and so the fitting of deformable models is problematic.

Effect of the presence or absence of J chain on expression of recombinant anti-Kell immunoglobulin M.

The aim of this study was to determine the effect of expressing a recombinant anti-Kell immunoglobulin (Ig) M from two cell lines, CH0 and NS0, on its ability to function as a diagnostic antibody. As a polymeric immunoglobulin, IgM is able to directly agglutinate red blood cells (RBCs), making it a useful blood grouping reagent. To simplify expression, recombinant human IgM (rIgM) from NS0 (a mouse myeloma line) and CHO (Chinese hamster ovary line) cells was expressed in the absence of human J chain. Whereas NS0 expresses mouse J chain, rIgM expressed from CH0 cells lack J chain. Although the ability to polymerize resides within the tailpiece of IgM heavy chain, J chain can influence the polymeric state. This in turn could affect the ability of rIgM to bind its antigen. The variable region of the heavy chain of an anti-Kell IgG was grafted onto the constant region of human IgM and co-expressed with light chain derived from the same antibody. rIgM was purified from each cell line and the strength of direct agglutination assessed. Both cell lines produced polymeric rIgM that was able to specifically bind the target antigen and to directly agglutinate RBCs to the same degree. The presence or absence of J chain did not affect the ability of the rIgM to bind the Kell antigen or the strength of agglutination. The presence of J chain is not required for the production of a functional rIgM for use as a diagnostic reagent. CHO and NS0 lines are both suitable for production of such a reagent.

Production of soluble recombinant proteins with Kell, Duffy and Lutheran blood group antigen activity, and their use in screening human sera for Kell, Duffy and Lutheran antibodies.

The aim of this study was to show that soluble recombinant (sr) proteins can mimic blood group antigens and be used to screen human sera for blood-group-specific antibodies. The blood of all pregnant women and pretransfusion patients should be screened for blood-group-specific antibodies to identify and monitor pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN), and to prevent haemolytic transfusion reactions. Current antibody screening and identification methods use human red blood cell panels, which can complicate antibody identification if more than one antibody specificity is present. COS-7 cells were transfected to produce sr forms of the extracellular domains of the red blood cell membrane proteins that express Kell, Duffy or Lutheran blood group antigens. These sr proteins were used to screen for and identify anti-Kell, anti-Duffy or anti-Lutheran blood-group-specific allo-antibodies in human sera by haemagglutination inhibition and in solid-phase enzyme-linked immunosorbent assays (ELISAs). There is a positive correlation (correlation coefficient 0.605, P value 0.002) between antibody titre by standard indirect antiglobulin test (IAT) and signal intensity in the ELISA test. This work shows that sr proteins can mimic blood group antigens and react with human allogeneic antibodies, and that such proteins could be used to develop solid-phase, high-throughput blood group antibody screening and identification platforms.

Can dynamic susceptibility contrast magnetic resonance imaging perfusion data be analyzed using a model based on directional flow?

PURPOSE: To examine the implications of a physiological model of cerebral blood that uses the contradictory assumption that blood flow in all voxels of DSCE-MRI data sets is directional in nature. Analysis of dynamic susceptibility contrast-enhanced magnetic resonance imaging (DSCE-MRI) uses techniques based on indicator dilution theory. Underlying this approach is an assumption that blood flow through pixels of gray and white matter is entirely random in direction. MATERIALS AND METHODS: We have used a directional flow model to estimate theoretical blood flow velocities that would be observed through normal cerebral tissues. Estimates of flow velocities from individual pixels were made by measuring the mean transit time for net flow (nMTT). Measurements of nMTT were made for each voxel by estimating the mean difference in contrast arrival time between each of the adjacent six voxels. RESULTS: Examination of the spatial distribution of contrast arrival time from DSCE-MRI data sets in normal volunteers demonstrated clear evidence of directional flow both in large vessels and in gray and white matter. The mean velocities of blood flow in gray and white matter in 12 normal volunteers were 0.25 +/- 0.013 and 0.21 +/- 0.014 cm/second, respectively, compared to predicted values of 0.25 and 0.18 cm/second. These values give measured nMTT for a 1-mm isotropic voxel of gray and white matter of 0.45 +/- 0.12 and 0.52 +/- 0.11 seconds, respectively, compared to predicted values of 0.47 and 0.55 seconds. CONCLUSION: A directional model of blood flow provides an alternative approach to the calculation of cerebral blood flow from (CBF) DSCE-MRI data.

Relationship between gravimetric wear and particle generation in hip simulators: conventional compared with cross-linked polyethylene.

Hip-simulator studies have shown reduced gravimetric wear rates for inert-gas gamma-irradiated ultra-high molecular weight polyethylene when compared with conventional ethylene-oxide-sterilized ultra-high molecular weight polyethylene. Analysis shows a greater number of particles generated from inert-gas gamma-irradiated ultra-high molecular weight polyethylene. This study was undertaken to examine particle-generation rates of polyethylene with different levels of cross-linking and to correlate them with gravimetric wear data. Particle-generation rates did not correlate with gravimetric wear rates. Particle analysis should be performed to predict the in vivo behavior of bearing surface materials. Cross-linked ultra-high molecular weight polyethylene subjected to 10 Mrad (100,000 Gy) of gamma irradiation generated significantly fewer particles than ethylene-oxide-sterilized ultra-high molecular weight polyethylene; it also demonstrated a 96% reduction in the volume of particles.

An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway.

The B cell activating factor BAFF (BlyS/TALL-1/zTNF4) is a tumor necrosis factor (TNF)-related ligand that promotes B cell survival and binds to three receptors (BCMA, TACI, and the recently described BAFF-R). Here we report an absolute requirement for BAFF in normal B cell development. Examination of secondary lymphoid organs from BAFF-deficient mice revealed an almost complete loss of follicular and marginal zone B lymphocytes. In contrast, mice lacking BCMA had normal-appearing B lymphocyte compartments. BAFF therefore plays a crucial role in B cell development and can function through receptors other than BCMA.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

Mechanisms of granulocytosis in the absence of CD18.

Genetic deficiency in CD18 leads to disease characterized by myeloid hyperplasia, including profound granulocytosis and splenomegaly. Myeloid hyperplasia could directly result from the disruption of CD18 functions essential to granulopoiesis or basal leukocyte trafficking. Alternatively, myeloid hyperplasia could be reactive in nature, due to disruption of essential roles of CD18 in leukocyte responses to microbial challenge. To distinguish between these mechanisms, the hematopoietic systems of lethally irradiated wild-type (WT) mice were reconstituted with either WT fetal liver cells or CD18-deficient fetal liver cells, or an equal mixture of both types of cells. Granulocytosis and splenomegaly developed in mice that received CD18-deficient fetal liver cells. Splenomegaly was prevented and granulocytosis was inhibited by more than 95% in mice that had received both CD18-deficient and WT fetal liver cells, suggesting that myeloid hyperplasia was largely reactive in nature. Consistent with this postulate, the circulating life spans in the blood and the fraction of neutrophils that incorporated BrdU in the bone marrow were not increased for CD18-deficient neutrophils compared with the WT. However, these animals did develop mild granulocytosis compared with mice reconstituted with WT cells alone, and a higher percentage of CD18-deficient leukocytes were neutrophils compared with the WT leukocytes. These observations suggest that the granulocytosis observed in the absence of CD18 occurs through at least 2 mechanisms: one that is dramatically improved by the presence of WT cells, likely reactive in nature, and a second that is independent of the WT hematopoietic cells, involving an alteration in the lineage distribution of blood leukocytes.

Pitfalls in surgical decision-making during mitral valve repair.

Mitral valve repair has become the mainstay of surgical treatment for mitral valvular regurgitation. Surgeons in North America were relatively slow to adopt the various repair techniques, perhaps because rheumatic heart disease was less common, and the initial experiences with large numbers of repairs in Europe dealt largely with rheumatic disease. Subsequent experience, however, has clearly shown that patients with degenerative mitral valve disease can expect very durable repairs, and that most such patients have relatively simple pathologic conditions. The potential for repair, with a lack of need for long-term anticoagulation, has led to earlier surgical intervention. Still, mitral valve repair is far more complex than mitral valve replacement and must be accompanied by careful intraoperative decision making. Pitfalls exist that are different from those that accompany replacement. In this article, we examine some of the more common problems, their identification, and, hopefully, ways to avoid them.

Monoclonal anti-D for immunoprophylaxis.

Routine antenatal prophylaxis with anti-D has become accepted as desirable, but concerns have been expressed about the adequacies of supply and safety of polyclonal anti-D. Human monoclonal anti-D has been produced using Epstein-Barr virus (EBV)-transformed peripheral B cells, sometimes coupled with fusions to myeloma cell lines. More recently, molecular biology techniques have been used to produce human monoclonal anti-D in a variety of different ways. Many monoclonal antibodies (mAbs) have been characterized for fine specificity and in vitro functional activity in International Workshops. Two mAbs have been shown to cause red cell clearance and immunosuppression in male volunteers. Considerations for the future development of monoclonal anti-D for prophylactic use are reviewed.

Monoclonal antibodies to Rh D--development and uses.

Monoclonal anti-D has proved impossible to make in rodent systems. Human monoclonal anti-D has been produced using EBV transformed peripheral B cells, coupled with fusions to myeloma cell lines. More recently molecular biology techniques have been used to produce monoclonal anti-D. The range of monoclonal anti-D produced is considered. The selection of monoclonal anti-D for use as blood grouping reagents for typing donors and recipients is reviewed--all types of D positive should be typed as positive when donors are considered. However, DVI patients should be typed as D negative. Considerations for the development of monoclonal anti-D for prophylactic use are reviewed.

Site directed mutagenesis of the human Rh D antigen: molecular basis of D epitopes.

Previous attempts to define the molecular configuration of D epitopes has been confined to the analysis of the serological profile and Rh D molecular structure of partial D phenotypes. There are numerous drawbacks in this approach, most fundamental of which is that with the exception of RoHar, partial D phenotypes are defined by the loss of D epitope expression, and is thus difficult to directly correlate a specific amino acid to a particular D epitope. Furthermore, most partial D phenotypes are associated with multiple amino acid changes in the mutant Rh protein species associated with partial D expression. In our study we have applied site directed mutagenesis to introduce RhD amino acids in a stepwise manner to a Rh cE cDNA. This cDNA was introduced into K562 cells using retroviral mediated gene delivery, and D epitope expression analysed by flow cytometry. Our study provides evidence for at least six different epitope clusters on the external face of the Rh D protein. The relative predicted positions of these epitope clusters has resulted in us presenting a model for the hypothetical arrangement of external Rh D protein loops.

Epitopes on Rh proteins.

Analyses of the reactions of monoclonal anti-D with Rh D variant red cells have shown that there are at least 24 different epitopes of the Rh D antigen. Similar studies Of Rh E variant red cells with monoclonal anti-E indicate that there are at least 4 epitopes of the Rh E antigen. The relation of these serologically defined epitopes to the structure of the Rh proteins is reviewed. Most epitopes are discontinuous, with critical residues present in different loops of the proteins.

Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2.

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome-microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.