Functional factor VIII made with von Willebrand factor at high levels in transgenic milk.

BACKGROUND: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. OBJECTIVES: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. METHODS: Transgenic mice were made with a mammary-specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, western blot, and one-stage and two-stage clotting assays. The hemostatic activity of immunoaffinity-enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. RESULTS AND CONCLUSIONS: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single-chain molecule that retained high specific activity, similar to therapeutic-grade FVIII. 226/N6 had > 450-fold higher IU mL(-1) than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle for the study of expression of 226/N6 and perhaps other single-chain bioengineered rFVIIIs in the milk of transgenic livestock.

In vitro development of bovine embryos cultured with stem cell factor or insulin-like growth factor-I following IVF with semen of two bulls having different field fertility.

The usefulness of IVF as a potential tool to evaluate the field fertility of bulls is equivocal and growth factor addition to culture media research is needed to delineate components needed for providing defined environments for embryos. The overall aim was to evaluate the in vitro development of embryos derived using a serum supplemented and serum-free production systems and semen from two bulls of different field fertility. The study was conducted to determine the combinatorial effect of stem cell factor (SCF) and/or insulin-like growth factor-I (IGF-I) in culture on subsequent embryo development in cattle. Oocytes were aspirated separately from >or=3 to <3mm follicles to test different follicle size populations and were matured in TCM-199 supplemented with LH, FSH, estradiol and BSA (Fraction V). Matured oocytes were fertilized in BSA supplemented synthetic oviductal fluid (SOF)-IVF medium. Presumptive zygotes were cultured for 8d (in humidified 5% CO(2) at 38.5 degrees C) in BSA supplemented SOF-in vitro culture (IVC) medium. SOF-IVC medium was supplemented with fetal bovine serum (4%), IGF-I (100ng/mL), SCF (50ng/mL) or IGF-I (100ng/mL)+SCF (50ng/mL). The development competence of embryos did not differ between the bulls and among the culture environments. Nevertheless, there was an effect of follicle size on cleavage rate (P<0.05) and a greater cleavage rate resulted from oocytes aspirated from >or=3mm follicles (71.0+/-1.5%) compared to those collected from <3mm follicles (64.8+/-1.6%). The overall cleavage rate (%); blastocyst formation (%); and expanded/hatched blastocyst formation (%) were 68.2+/-1.5 and 67.7+/-1.7; 29.4+/-1.4 and 28.6+/-1.5; and 18.6+/-1.2 and 18.5+/-1.1, respectively, for the bull of above and below average field fertility. The results indicate that follicle size for oocyte aspiration is effective for determining IVC success and that IVF may not discriminate among bulls of different field fertility.

Effect of droplet vitrification on development competence, actin cytoskeletal integrity and gene expression in in vitro cultured mouse embryos.

The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol (VS-1) and ethylene glycol plus DMSO (VS-2) and thawed by directly placing the vitrified drop into 0.3M sucrose solution at 37 degrees C. High recovery (93-99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/or in vitro culture. The expression pattern of Bax, Bcl2 and p53 genes was altered (P<0.05) in vitrified zygotes and 2-cell embryos, but not in morulae. Although a large proportion of the vitrified zygotes (59.5+/-4.4% in VS-1 and 57.9+/-4.5% in VS-2; mean+/-S.E.M.) and 2-cell embryos (63.1+/-4.4% in VS-1 and 59.2+/-4.3% in VS-2) developed into blastocysts, development of control embryos (70.2+/-5.0% of zygotes and 75.5+/-4.4% of 2-cell embryos) into blastocysts was higher (P<0.05). In contrast, development of the control and vitrified morulae into blastocysts (more than 85%) was similar. We concluded that the modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos.

Gene expression and development of mouse zygotes following droplet vitrification.

The concept of ultra-rapid vitrification has emerged in recent years; the accelerated cooling rate reduced injury attributed to cryopreservation and improved post-freezing developmental competence of vitrified oocytes and embryos. The objectives of the present study were to develop a simple and effective ultra-rapid vitrification method (droplet vitrification) and evaluate its effects on post-thaw development and apoptosis-related gene expression in mouse zygotes. Presumptive zygotes were equilibrated for 3 min in equilibration medium and washed 3 times in vitrification solution. A drop (5 microL) of vitrification solution containing 10-12 embryos was placed directly onto surface of liquid nitrogen, with additional liquid nitrogen poured over the drop. For thawing and cryoprotectant removal, vitrified drops were put into dilution medium for 3 min, followed by M2 medium for 5 min. Although cleavage rate did not differ significantly among the control (90.8+/-2.8%; mean+/-S.E.M.), toxicity control (83.5+/-3.2%), and vitrified (86.2+/-3.1%) zygotes, rates of blastocyst and hatched blastocyst formation were lower (P<0.01) in vitrified zygotes (49.7+/-4.7% and 36.0+/-4.7%) and toxicity controls (47.3+/-4.6% and 40.3+/-4.6%) compared with controls (65.5+/-4.1% and 54.2+/-4.3%). Exposure of zygotes to vitrification solution, as well as the vitrification process, down-regulated the expression of Bax, Bcl2, and p53 genes in blastocysts. Although droplet vitrification was efficient and easy, it altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes.

Development of murine embryos following electroporation.

PURPOSE: The objective was to establish the parameters for reversible electroporation of murine embryos. METHODS: In Trial 1, murine presumptive zygotes received an electrical pulse of 5, 10, or 20-micros duration, and one of five voltages (100, 200, 250, 300, or 400 V). In Trial 2, embryo orientation within the electroporation chamber was evaluated with 250 or 400 V at a pulse period of 10 micros. RESULTS: Presumptive zygotes that received 400 V at each pulse length and zygotes exposed to 20 micros at each voltage had the lowest embryonic development (P < 0.05). Presumptive zygotes that received 250 V had higher development compared to 400 V, irrespective of orientation (P < 0.01), but development was lower than the controls (P < 0.01). CONCLUSIONS: Electrical stimulation of presumptive zygotes can have a detrimental impact on early embryo development, but low amounts of stimulation may allow for potential gene transfer in transgenic experimentation.

Nocturnal blood pressure predicts left ventricular mass index in normotensive elderly.

Blood pressure (BP) predictors of left ventricular mass index (LVMI) were studied in 40 healthy normotensive (71.4 +/- 4.4 years) and 31 hypertensive (73.5 +/- 4.8 years) elderly community-dwelling subjects using short-axis cardiac cine magnetic resonance imaging and 24-h ambulatory BP monitoring. Mean night-time BPs were calculated from the average of readings during sleep and mean daytime BPs were calculated from the remaining recordings. The hypertensive subjects were all receiving anti-hypertensive therapy with angiotensin-converting enzyme (ACE) inhibitors, calcium-channel blockers, beta-blockers or diuretics. Nocturnal systolic BP was a strong predictor of LVMI in both normotensive (beta = 0.38, p = 0.02) and treated hypertensive (beta = 0.39, p = 0.03) subjects. By contrast, daytime systolic BP was a weaker predictor of LVMI in the treated hypertensives (beta = 0.36, p = 0.04) and did not predict LVMI in the normal subjects (beta = 0.27, NS). Nocturnal BP may partly explain the increase in LVMI with ageing in subjects thought to be normotensive on the basis of daytime clinic BP recordings.

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland.

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct.

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 +/- 0.1) than for grade B (0.8 +/- 0.1), C (0.6 +/- 0.1), or D (0.3 +/- 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos.

Reproducibility study of left ventricular measurements with breath-hold cine MRI using a semiautomated volumetric image analysis program.

The reproducibility of a semiautomated method of volumetric analysis allowing estimates of left ventricular (LV) parameters in approximately 5 minutes of analysis time is reported. Twenty normal volunteers underwent cine breath-hold cardiac MRI on two occasions with two observers using this new semiautomated method to estimate LV parameters. Reproducibility of this technique was comparable to published data with a variability of less than approximately 10% for all LV parameters calculated. Using this technique, the 95% confidence limits for change for left ventricular end diastolic volume (LVEDV) = +/-15 ml, left ventricular end systolic volume (LVESV) = +/-8 ml, LV mass = +/-24 g, and left ventricular ejection fraction (LVEF) = +/-6%. This new method also compared favorably to established manual methods. This new method permits estimation of LV parameters with acceptable reproducibility in a time that may permit routine quantitation of cardiac MR studies.

Current progress in the production of recombinant human fibrinogen in the milk of transgenic animals.

The mammary gland of transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein. While the mammary gland appears to be able to secrete fully assembled recombinant human fibrinogen (rhfib) at 0.1 to 5 g/l levels, some unassembled rhfib chains are also secreted. Presently, the relationship between unassembled rhfib and the coordinated translation of each nascent rhfib polypeptide in the mammary epithelia is unknown. The secretion of fully and partially assembled rhfib is widely variable among mammalian cell lines and where previously no cell line has been shown to secrete beta chain alone. We have observed that mammary epithelia can secrete B beta chain into milk as well as immature forms of other recombinant proteins, suggesting it likely uses a different secretory pathway than does the liver. This difference in secretory behavior is possibly due to the natural design of milk, where the precise regulation of post translational modifications and intracellular pools of nascent polypeptides needed to achieve fibrinogen assembly may be less important to fulfill the nutritional function of most milk proteins.

Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C.

The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100-1800 micrograms ml-1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100-400 micrograms ml-1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein.

Detection of postoperative deep-venous thrombosis using technetium-99m-labeled tissue plasminogen activator.

UNLABELLED: The current noninvasive methods of deep-venous thrombosis (DVT) detection in the asymptomatic patient are sufficiently inaccurate so as to preclude their routine use. This present study reports the accuracy of scintigraphic scanning with 99mTc-rt-PA in asymptomatic postoperative patients using contrast venography as the gold standard. METHODS: Fifty-three consecutive postarthroplasty patients (30 THR, 23 TKR) (16 women, 37 men; mean age 71 yr; range 52-85 yr) underwent scintigraphic scanning with 99mTc-rt-PA and contrast venography, on the operated leg, in order to assess the accuracy of this new technique in these asymptomatic patients. RESULTS: Eighty-four segments were of diagnostic quality on contrast venography. Of the 15 thrombosed segments, 14 had positive scans. In the 69 nonthrombosed segments, 63 had negative scans. Thus, scintigraphic scanning with 99mTc-rt-PA had a sensitivity of 93% and a specificity of 91%. CONCLUSION: This study demonstrated that scintigraphic scanning with modified 99mTc-rt-PA is accurate in the detection of DVT in patients undergoing total hip or total knee arthroplasty.

Measurement of periprosthetic bone density in hip arthroplasty using dual-energy x-ray absorptiometry. Reproducibility of measurements.

To define the precision (reproducibility) of measurement of periprosthetic bone mineral density and bone mineral content, dual-energy x-ray absorptiometry scans were obtained on 45 randomly selected patients who had had a unilateral total hip arthroplasty within the previous 3 years. The coefficients of variation of the bone mineral density in the proximal Gruen zones were 5.0 and 5.3%, corresponding to errors of 0.07 and 0.11 g/cm2. The coefficients of variation of the bone mineral density for the distal zones averaged 2.8%, with an error of 0.08 g/cm2. The coefficients of variation of the bone mineral content were 4.8 and 2.9% for the proximal and distal zones. The contralateral femur was also scanned in 32 of the patients. For the contralateral femur bone mineral density, the coefficients of variation were 5.0% for the proximal zones and 4.8% for the distal zones. The bone mineral content was 6.0% for the contralateral regions. These results imply that differences in bone mineral density greater than 0.16 g/cm2 (2 standard errors) can be reliably measured. Dual-energy x-ray absorptiometry therefore provides a highly reproducible technique for quantitatively monitoring the changes in bone density that occur after total hip arthroplasty.

Technetium-99m-modified recombinant tissue plasminogen activator to detect deep venous thrombosis.

UNLABELLED: We report a method for deep venous thrombosis (DVT) detection which uses 99mTc-labeled modified recombinant tissue plasminogen activator (rt-PA) scintigraphy. A Phase III clinical trial was performed on 79 patients with suspected DVT. METHODS: The plasminogen binding site of rt-PA was permanently inhibited without inactivating the fibrin binding site. The modified molecule was radiolabeled with 99mTc. Scintigraphy was performed and the results were compared to those of contrast venography. RESULTS: Of 14 thrombosed proximal segments, 13 had positive scans; in the 53 nonthrombosed proximal segments, 49 had negative scans. In proximal vein thrombosis, rt-PA scintigraphy had a sensitivity of 93% and a specificity of 92%. Of the 36 thrombosed calf vein segments, 31 had positive scans; in the 30 nonthrombosed calf segments, 28 had negative scans. In calf vein thrombosis, scanning has a sensitivity of 86% and a specificity of 93%. CONCLUSIONS: Scintigraphic scanning with this 99mTc modified rt-PA permits accurate detection of thrombus in both proximal and calf veins in patients with clinically suspected DVT. The technique detects both fresh and aged thrombi and is unaffected by heparin administration. Further study in other patient groups is needed to define the overall clinical utility.

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures.

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.

Stress protection due to external fixation.

Bone is sensitive to mechanical influences. The presence of an orthopaedic device will impose constraints on the mechanical environment that may influence subsequent remodelling and repair. An Oxford External Fixator was applied to six intact ovine tibiae. The strains engendered during normal walking were then recorded from strain gauges applied to the mid-shaft of the bone. The fixator configuration was then altered such that the intact fixator bar connecting the pins was replaced with a sectioned version that did not permit load transfer through the fixator, and the strain environment re-recorded. The peak strains recorded with the intact bar were significantly lower than those recorded with the sectioned bar. This showed that the use of a fixator with an intact bar resulted in significant stress protection of the underlying bone. A fixator was then applied to both the right and left tibiae of a further six animals and the resulting strain environment and corresponding remodelling response was observed over 16 weeks. In each case one fixator was configured with an intact bar (the stress protected limb), whilst the other utilised a sectioned bar (the unprotected limb). The results showed that over this period the bone mineral content fell by 9% in the stress protected limb compared to the unprotected limp. Quantitative assessment of the bones showed that this bone loss occurred as a direct consequence of resorption on the periosteal and endosteal surfaces. In addition, strain recordings at week 16 showed that the fixator was still stress protecting the tibia.

Scintigraphic assessment of colonic function during defaecation.

The aim of this study was to develop a method by which rectal and colonic activity could be examined during defaecation under physiological conditions, in order to evaluate whether the colon plays a role in defaecation. Subjects presented to the Nuclear Medicine department on the day following ingestion of oral In-111 labelled DTPA, when they developed the normal urge to defaecate. Defaecation took place in a private room while dynamic scintigraphy of the rectum and colon was recorded. Fourteen subjects were studied (8 normal subjects, 4 with constipation, 2 with irritable bowel syndrome). In 13 subjects the left colon was visualized during defaecation and emptying was clearly observed in 12. The right colon was visualised in 11 subjects and emptying was seen in 7. Mean percentage segmental evacuation was right colon 20%, left colon 32% and rectum 66%. Colonic emptying occurs during defaecation, which is not a process of rectal evacuation only. This has implications for the understanding of the pathophysiology of obstructed defaecation.

Incidence of pulmonary embolism in single segmental mismatch on lung scanning.

UNLABELLED: Controversy exists as to whether patients with single segmental mismatch (SSM) on a ventilation/perfusion (VQ) lung scan should be given a low or an intermediate probability of pulmonary embolism (PE). METHODS: Pulmonary angiography was used to evaluate the incidence of PE in SSM at the authors' institution. From January 1991 to January 1993, 1449 VQ scans were performed. RESULTS: With modified Biello criteria, 283 were high probability; 628, low probability; 273, normal; and 273, intermediate probability. Of the intermediate probability scans, 61 had SSM. Forty of these patients underwent pulmonary angiography. Twelve patients had PE in the area of the SSM, giving an incidence of PE of 30%. The risk of PE in SSM in the different lung regions was also analyzed. Twenty-three SSM were in the bases of the lung with a 22% incidence of PE; 17 SSM were either in the midzone or apex with a 41% incidence of PE (p = not significant). CONCLUSION: SSM carries a 30% risk of PE. Accordingly, SSM should be given an intermediate probability of PE and not a low probability of PE.

Influence of aging on oral-pharyngeal bolus transit and clearance during swallowing: scintigraphic study.

The aim of the study was to determine the influence of normal aging on regional transit and the efficiency of bolus clearance during the oral and pharyngeal phases of swallowing. We compared scintigraphically derived oral-pharyngeal transit times and isotope clearance during swallowing in 21 healthy aged volunteers (mean age 68 +/- 8 yr) and 9 young controls (mean age 28 +/- 7.5 yr). Subjects swallowed 5- and 10-ml water boluses mixed with 30 MBq 99mtechnetium tin colloid. Oral and pharyngeal transit times, pharyngeal clearance time, and postswallow residual counts in each region were derived from time-activity curves. Pharyngeal residual counts were significantly greater in the aged than in controls (P = 0.0008), but age did not influence oral residual. Aging significantly prolonged oral transit time (P = 0.02), pharyngeal transit time (P = 0.0004), and pharyngeal clearance time (P = 0.0001). We conclude that normal impairs the efficiency of pharyngeal clearance during swallowing, prolongs scintigraphic measures of oral-pharyngeal transit, and increases the exposure time of the glottis to the swallowed bolus.