Rapid-sequence tandem transplant for children with high-risk neuroblastoma.

BACKGROUND: The majority of patients with high risk neuroblastoma (NB) still relapse. PROCEDURE: We designed a Phase II trial for children with advanced NB utilizing a program of induction chemotherapy followed by tandem high-dose chemoradiotherapy with stem cell rescue (HDC/SCR) in rapid sequence. Fifty-five patients were evaluable, ages 1-14 years, and 97 cycles of HDC/SCR have been completed to date. Pheresis was possible for every patient, despite their young age, with an average of 7.2 x 10(6) CD34+ cells/kg available to support each HDC/SCR cycle. RESULTS: Engraftment was rapid, with median time to neutrophil engraftment of 11 days. Five patients who completed the first HDC course did not complete the second and there were four toxic deaths. With a median follow-up of 24 months from diagnosis, 38 of 55 patients (3-year EFS 59%) remain event-free. A subset of the patients received stem cells purged by CD34 selection. The engraftment and EFS of these patients are similar to the overall group. CONCLUSION: This work demonstrates that a tandem transplant regimen for high-risk NB is a feasible treatment strategy in children and may improve disease-free survival.

Tandem high-dose therapy in rapid sequence for children with high-risk neuroblastoma.

PURPOSE: Advances in chemotherapy and supportive care have slowly improved survival rates for patients with high-risk neuroblastoma. The focus of many of these chemotherapeutic advances has been dose intensification. In this phase II trial involving children with advanced neuroblastoma, we used a program of induction chemotherapy followed by tandem high-dose, myeloablative treatments (high-dose therapy) with stem-cell rescue (HDT/SCR) in rapid sequence. PATIENTS AND METHODS: Patients underwent induction chemotherapy during which peripheral-blood stem and progenitor cells were collected and local control measures undertaken. Patients then received tandem courses of HDT/SCR, 4 to 6 weeks apart. Thirty-nine patients (age 1 to 12 years) were assessable, and 70 cycles of HDT/SCR were completed. RESULTS: Pheresis was possible in the case of all patients, despite their young ages, with an average of 7.2 x 10(6) CD34(+) cells/kg available to support each cycle. Engraftment was rapid; median time to neutrophil engraftment was 11 days. Four patients who completed the first HDT course did not complete the second, and there were three deaths due to toxicity. With a median follow-up of 22 months (from diagnosis), 26 of 39 patients remained event-free. The 3-year event-free survival rate for these patients was 58%. CONCLUSION: A tandem HDT/SCR regimen for high-risk neuroblastoma is a feasible treatment strategy for children and may improve disease-free survival.

Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos.

This study examined the effects of incorporating an ovine oviducal oestrus-associated glycoprotein (oEGP) and amino acids, at the concentrations present in the ovine oviduct around the time of oestrus, on in vitro production and subsequent viability of bovine embryos. The first experiment compared the influence of ovine oviducal concentrations of amino acids with MEM and BME amino acids. There was no treatment effect on cleavage rate (74.9% vs. 75.5%), but there was a higher (P < 0.05) blastocyst yield (30.4 vs. 25.2) and a shorter time (P < 0.05) to blastocyst formation (7.16 +/- 0.64 vs. 7.27 +/- 0.56 days) following use of oviducal concentrations of amino acids. Experiment 2 examined the influence of oEGP in combination with each of the amino acid treatments. oEGP had no effect on cleavage or blastocyst yield within amino acid treatments. Day of blastocyst formation significantly influenced nuclei numbers (P < 0.001) with higher numbers being obtained on day 7 than on either day 6 or day 8. There was also a significant (P < 0.01) interaction between day of blastocyst formation and amino acid treatment on blastocyst nuclei numbers. The third experiment studied the effects of the amino acid treatments on embryo viability. There was no effect of amino acid treatment of embryos on pregnancy rates (34.5 vs. 44.4%) following transfer of days 6 and 7 blastocysts to synchronized recipients. oEGP did not influence any of the parameters of bovine embryo development that were measured, suggesting that effects of this protein observed on ovine embryos are species specific. It is concluded that ovine oviducal amino acid concentrations are beneficial to blastocyst development in vitro but do not have any further beneficial effect following transfer of blastocysts to recipients.

Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice.

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-ace B gene-ovine growth hormone (GH) gene (3' GH sequence) construct was fused to the ovine, MT-Ia promoter-ace A gene-ovine GH gene (3' GH sequence). Therefore, in this single DNA sequence, both ace A and ace B are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of the ace B-ace A gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressed Escherichia coli cells.

Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations.

The effects of supplementing synthetic oviductal fluid (SOF) with amino acids, at oviductal fluid concentrations, on the development of ovine in vitro-matured/in vitro-fertilized embryos was examined in three experiments. In the first, embryo development in SOF, SOF + 2% human serum (HS), SOF + 20% HS, and SOF + BSA, with and without amino acid supplementation, was examined. Development of zygotes to the blastocyst and hatching blastocyst stages was highest in medium containing 20% HS (64.8% and 54.4%, respectively) irrespective of amino acid supplementation. However, supplementation was significantly beneficial in all other media, with up to 42.1% of zygotes developing into hatching blastocysts. In these media, supplementation also significantly increased the mean number of nuclei per newly formed blastocyst (up to a mean of 70.8) and reduced the time during which blastocysts formed. Experiment 2 was an examination of the effect on embryo development of three amino acid preparations (oviduct amino acid concentrations vs. Eagle's Basal Medium (BME) essential + Minimum Essential Medium (MEM) nonessential vs. MEM essential + MEM nonessential concentrations) and the presence or absence of BSA. Both the amino acid and BSA treatments significantly influenced the percentage of zygotes that developed to the hatching blastocyst stage but not to the blastocyst stage. The preferred medium contained amino acids at oviductal fluid concentrations and BSA (54.5% hatching rate). The amino acid treatments did not significantly influence the mean number of nuclei per newly formed blastocyst, but the addition of BSA had a significant effect (70.7 +/- 1.14 vs. 75.7 +/- 1.13). In experiment 3, embryo development to Day 13 was examined after culture in SOF containing amino acids at oviductal fluid concentrations. Embryos were cultured in the presence of either BSA, polyvinyl alcohol (PVA), or no additional supplement and were transferred to recipient ewes on either Day 0 (after in vitro fertilization), 3, or 5. The addition of BSA or PVA had no significant effect, but significantly more embryos developed to Day 13 following transfer on Day 0 (60.0%) than on either Day 3 or 5 (overall 45.4%). It is concluded that SOF containing oviductal fluid concentrations of amino acids 1) facilitates the development of a high percentage (57.5%) of blastocysts, 2) improves embryo morphology compared with that observed in medium containing HS, 3) significantly improves hatching rates compared with those obtained in SOF containing commercially available preparations of amino acids, and 4) produces embryos with relatively high levels of viability to Day 13 of pregnancy.

Cloning and sequencing of a cDNA encoding an ovine oestrus-associated oviducal protein.

Ovine oestrus-associated oviducal glycoprotein (oEGP) is synthesized and secreted specifically by the ampullary region of the ovine oviduct during the peri-ovulatory stages of the oestrous cycle. A cDNA that encodes oEGP was isolated and sequenced. Isolation of oEGP was achieved using the polymerase chain reaction (PCR) with primers based on a bovine oestrus-associated oviducal glycoprotein cDNA (bOGP) sequence. A 1599-bp cDNA encodes, in part, a deduced 519-amino acid sequence of mature protein which carries two potential N-linked glycosylation sites. The deduced amino acid sequence is more than 95% identical to that of bOGP and more than 74% identical to the first 491 amino acids of human oestrogen-dependent oviducal glycoprotein (hOGP). Northern blot hybridizations of RNA from several sheep tissues detected mRNA (2.4 kb) only in an ampulla oviduct sample.

The commercial and agricultural applications of animal transgenesis.

The potential for commercial application of transgenic technologies in domestic animals is discussed in relation to the areas where a significant impact on agriculture might be expected. These are the endocrine system, novel biochemical pathways, structural proteins of milk and of textile fibers, and the immune system. Manipulation of the endocrine system has been investigated for some years and it is clear that very accurate control is needed over gene expression if this approach is to prove commercially useful. The area most advanced in commercial application is the production of high-value pharmaceutical proteins in the mammary glands of domestic animals. Other applications that are discussed remain to be proven in larger animals despite being demonstrated laboratory test animals. These include a functional cysteine biosynthetic pathway and a functional glyoxylate cycle transferred from bacteria to mice, and alterations to the proteins of hair that change the physical properties of the resultant fibers. Research is also actively directed toward novel approaches for providing domestic animals with resistance to insects.

Oviduct proteins in fertilization and early embryo development.

The oviduct controls the environment in which the gametes are transported and fuse, and in which embryonic development begins. The ultrastructural topography of the ampulla and isthmus is similar, consisting of ciliated and secretory cells, but a different array of proteins is secreted by each segment along with various serum components. Amino acids are selectively secreted by the oviduct; these amino acids probably interact with the gametes or embryo to facilitate the processes of fertilization and development. An oviduct-specific glycoprotein is synthesized by the ampulla of sheep and cattle in response to oestrogen and secreted mainly from day-1 to day 3 of the ovarian cycle. This oestrus-associated glycoprotein (EGP) has a variable molecular mass of 80-97 kDa and a pI value ranging from 4.7 to 5.5. The bovine (b) and ovine (o) EGP genes are 95.5% identical and consist of 1560 base pairs encoding 519 amino acids containing one N-linked and several O-linked glycosylation sites. The terminal glycosides are N-acetylglucosamine and galactose-N-acetylgalactosamine for bEGP, and fucose, galactose and sialic acid residues are also identified for oEGP. EGP binds to zona pellucida and blastomere membranes, but evidence for EGP binding to sperm membranes is equivocal. After in vitro fertilizaton the proportion of sheep oocytes cleaving was increased in the presence of oEGP, but when single-cell embryos were cultured with oEGP, these cleavage rates were reduced. In addition, consistent positive effects of oEGP were observed on blastocyst formation. Elaboration of the mechanism of synthesis of EGP, its action and its role in fertilization and embryo development is important for our understanding of the events of early pregnancy.

Co-culture, oviduct secretion and the function of oviduct-specific glycoproteins.

Co-cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts. Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development. Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media. Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media. These glycoproteins are induced by oestrogen in vivo but not in vitro. It is our contention that co-cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells.

Anaesthesia for abdominal vascular surgery in patients with coronary artery disease (CAD), Part I: Isoflurane produces dose-dependent coronary vasodilation.

The effects of anaesthesia for major abdominal vascular surgery on coronary flow regulation and mechanisms of myocardial ischaemia were studied in 56 patients with CAD, using a randomized, partly double-blinded protocol. After induction with fentanyl (3 micrograms.kg-1) and thiopentone (2-4 mg.kg-1) and tracheal intubation, principal anaesthetics were nitrous oxide/oxygen (60/40) with isoflurane (n = 20), halothane (n = 19) or fentanyl (15-20 micrograms.kg-1) (n = 17). Conventional invasive techniques and coronary venous retrograde thermodilution were used to assess systemic and coronary haemodynamics. Coronary vascular resistance was estimated from myocardial oxygen extraction. Myocardial ischaemia was diagnosed by 12-lead ECG and/or anterior wall motion abnormalities by cardiokymography and/or myocardial lactate production. When adjustment of anaesthetic dose was insufficient for haemodynamic control, i.v. phenylephrine and nitroglycerine were administered to treat hypotension and hypertension or cardiac failure respectively. Measurements were performed at four specific intervals; awake, before surgery and 10 and 30 min after abdominal incision. Comparable changes of systemic haemodynamics and myocardial oxygen consumption were observed in the three groups. Coronary vasodilation was evidenced in isoflurane patients only and was linearly dose-dependent (P < 0.001). Partial Least Squares Projections to Latent Structures modelling with cross validation confirmed this dose-dependency and ruled out a clinically measurable influence by intervention drugs or simultaneous systemic haemodynamic abnormalities. The incidence of myocardial ischaemia during anaesthesia and surgery was comparable in the three groups (35, 37 and 24%, respectively) and there was an association with systemic haemodynamic aberrations in 19 of the 27 ischaemic episodes. In contrast to ischaemic halothane and fentanyl patients, isoflurane patients with ischaemia had significantly lower myocardial oxygen extraction (P = 0.008 and P = 0.001, respectively), indicating that the oxygen extraction reserve was not utilized in a normal way during ischaemia.

Reproductive wastage in the androstenedione-immune ewe.

An experiment was carried out using 320 adult Merino ewes to examine the effects of immunization against an androstenedione human serum albumin conjugate (Fecundin) on ovulation rate, fertilization rate and embryo viability at days 2, 9 and 13-14 after fertilization. The ovulation rate of immunized ewes (2.19 +/- 0.06) was greater (P < 0.001) than that of control ewes (1.43 +/- 0.04). The recovery rate of embryos or of unfertilized oocytes on day 2 was reduced in immunized ewes, but fertilization rate of recovered oocytes was unaffected by immunization. The mean number of normal embryos per ewe pregnant (prolificacy) was higher and the proportion of ewes pregnant (fertility) was lower in immunized than in the control ewes. The distribution of the number of cells per embryo showed no differences in developmental age over the period of sampling, the majority of embryos at this time being at the two- to four-cell stage of development. At day 9 of pregnancy, blastocyst recovery rates were lower in immunized than in control ewes. About 90% of blastocysts recovered were developing normally in control ewes compared with 64% in immunized ewes. The majority of blastocysts recovered on day 9 had hatched from the zona pellucida prior to recovery (mean values were 94.2% and 87.8% for control and immunized groups, respectively). In control ewes single blastocysts were larger than twin blastocysts, but for the immunized ewes this difference was not significant. Both single blastocysts (P < 0.01) and twin blastocysts (P < 0.05) from immunized ewes were smaller than those from control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue distribution of bupivacaine enantiomers in sheep.

rac-Bupivacaine HCl was infused intravenously to constant arterial blood drug concentrations in sheep using a regimen of 4 mg/min for 15 min followed by 1 mg/min to 24 h. At 24 h, arterial blood was sampled, the animal was killed with a bolus of KCl solution, then rapidly dissected and samples were obtained from heart, brain, lung, kidney, liver, muscle, fat, gut, and rumen. Tissue:blood distribution coefficients for (+)-(R)-bupivacaine exceeded those of (-)-(S)-bupivacaine (P < 0.05) for heart, brain, lung, fat, gut, and rumen by an overall mean of 43%. Blood:plasma distribution coefficients of (-)-(S)-bupivacaine exceeded those of (+)-(R)-bupivacaine by a mean of 29% and this offset the tissue:blood distribution coefficients so that the previously significant enantioselective differences disappeared. It is concluded that although enantioselectivity of bupivacaine distribution is shown by the measured tissue:blood distribution coefficients, it is not shown when tissue:plasma water distribution coefficients are calculated, suggesting that there is no intrinsic difference between the bupivacaine enantiomers in tissue affinity. Sheep given fatal intravenous bolus doses of rac-bupivacaine had significantly greater concentrations of (+)-(R)-bupivacaine than (-)-(S)-bupivacaine in brain (P = 0.028) and ventricle (P = 0.036); these could augment the greater myocardial toxicity of this enantiomer found in vitro.

The commercial and agricultural applications of animal transgenesis.

The commercial potential for transgenesis techniques is substantial, particularly in the fields of animal and plant agriculture. This results from productivity being a function of genetic potential and interaction with the environment, but environmental factors being only partially subject to influence by the farmer. Thus, concentrating on genotype improvement becomes an important goal if substantial cumulative gains in productivity are to be made. Historically, the genetic potential associated with important animal production traits, such as wool growth, milk yield, and body growth, has been improved by selective breeding, whereby phenotypically superior animals are used as parental stock for following generations. The high quality of the domestic animals in use in farming today compared with those of earlier centuries is witness to the success of the approach, but nevertheless, the method has significant limitations that have frustrated animal breeders for many years. The complex genetic interactions that combine to produce a particular animal phenotype result in slow genetic gain, averaging at best about 1-3% per year. In addition, separating a desired production trait from one or more undesirable traits is often very difficult. However, the most important of these limitations is the inability to transfer genetic information between species, because of the biological barrier that prevents interspecific breeding.

Production of transgenic sheep.

The production of transgenic sheep has proven difficult compared to the mouse and lower animals. The work load is far greater and the rates of success far less by most criteria. However, the benefits to human and animal health and agricultural productivity are potentially enormous (Ward and Nancarrow, Chapter 5) and support for the continuation of the work is assured. Unfortunately, the low rate of transgenesis for sheep, at about 1% of injected, transferred embryos, means that investigation of the regulation of expression of the transgenes, their phenotypic effects, and optimization of the fusion gene constructs, all of utmost importance to the agricultural industry, can seldom be addressed. We know now that the mouse may not be a good model for the sheep, an example being the ovine metallothioneinovine growth hormone fusion gene, GH9, for which expression and phenotypic effects were quite different for sheep and mice. In sheep, pronuclear microinjection of several hundred copies of the foreign gene into embryos is the only published method used to regularly produce transgenics and it will be the standard by which future methods for incorporation of the transgene are judged.

Linear bone growth of oMT1a-oGH transgenic male mice.

Linear bone growth was studied in male mice possessing a controlled ovine metallothionein 1a promoter-ovine growth hormone (oMT1a-oGH) transgene. Transgene expression was activated at weaning by the addition of 25 mM zinc sulfate to drinking water; transgenic and control mice received the zinc supplementation. The ulna, humerus, and tibia were excised at 10-day intervals until 130 days from control and from mice hemizygous for oMT1a-oGH. Bones from mice overexpressing growth hormone (GH) were 11-20% longer than those from controls (P less than 0.01) at 130 days. Transgenic mice exhibited both an enhanced rate of bone growth and a growth period of greater duration, i.e., the ulna, tibia, and humerus from oMT1a-oGH mice grew at an accelerated rate for an additional 20-40 days relative to the same bones from control mice. The bones from both groups were characterized by isometric growth patterns. Genetic size scaling revealed that the observed differences in bone growth were directly related to the mature size of the bone, suggesting that the bones possess an inherent growth pattern that is followed even in the presence of elevated GH.

The genetic engineering of production traits in domestic animals.

The transfer of recombinant DNA by microinjection of embryo pronuclei provides a novel approach to the manipulation of production traits in domestic animals. In this review, several of the key areas currently under investigation are examined and their progress evaluated. These include the manipulation of the endocrine system by altered growth hormone genes and the modification of animal biochemistry by the introduction of the cysteine biosynthetic pathway and the glyoxylate cycle. The possibilities inherent in the alteration of structural proteins important to domestic animal productivity, and some ethical issues relevant to the release of modified animals are also considered. The experimental information obtained so far in the area indicates that transcriptional regulation of the genes and a thorough understanding of the physiological processes involved are both important factors in the practical application of the technique to the improvement of animal productivity.

The in vivo blood, fat and muscle concentrations of lignocaine and bupivacaine in the hindquarters of sheep.

1. A method was developed for sampling muscle and fat from the hindquarters of sheep undergoing spinal anaesthesia. The method was used to measure the concentrations of lignocaine and bupivacaine in the blood, muscle and fat of the hindquarters of sheep during and after 180 min constant-rate infusions of the drugs. 2. For both drugs the muscle drug concentrations were a relatively constant ratio of the simultaneous arterial blood drug concentrations during and after the infusion. 3. There was uptake of both lignocaine and bupivacaine into subcutaneous fat during the infusions. At the end of the infusion the ratio of the fat: arterial blood drug concentrations were 1.54 (SD = 0.57, n = 4) and 3.1 (SD = 1.4, n = 4) for lignocaine and bupivacaine, respectively. 4. The drug concentrations in fat declined relatively slowly after the infusion. The ratio of the fat: arterial blood drug concentrations 180 min after the end of the infusion was 21.5 (SD 4.0, n = 3) and for lignocaine, and 120 min after the end of the infusion was 9.54 (SD 5.2, n = 3) for bupivacaine. 5. It was concluded that the concentrations of lignocaine and bupivacaine in muscle were essentially in equilibrium with the arterial concentrations during and after the infusion. However, the concentrations of lignocaine and bupivacaine in fat were not in equilibrium with the arterial concentrations in the post-infusion period.

The potential of transgenic animals for improved agricultural productivity.

The techniques involved in the transfer of foreign DNA to domestic animals have advanced to the stage where transgenic animals that express foreign genes can be reliably produced, albeit still at low efficiency. This paper reviews the current status of some of the more important areas in agriculture where this technology is being applied. Numerous attempts have been made to modify the growth performance characteristics of domestic animals by the introduction of metallothionein/growth hormone fusion genes. A summary of our work with transgenic sheep is presented. The results demonstrate that the unregulated production of growth hormone in transgenic sheep reduces carcass fat, elevates metabolic rate and heat production, causes skeletal abnormalities and impairs survival. The introduction of new metabolic pathways to domestic animals offers an attractive approach to improved animal productivity. This paper summarises recent results of research directed towards the introduction of a cysteine biosynthetic pathway and the glyoxylate cycle to transgenic sheep. So far, the genes encoding the enzymes have been isolated and expressed both in cells in culture and in transgenic mice. The results of work currently in progress demonstrate that some modification of the fusion genes is required to enhance their expression in transgenic animals.