Search PubMed⌕ Search

Biomedical subjects

R Milner

Publications and source records attributed to R Milner.

At least 37 records · Page 2Linked to original sources
PubMed1999

A kinetic model for the homing and migration of prenatally transplanted marrow.

Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (10(6) LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.

Animals↗
PubMed1999

Mapping regions of the beta1 integrin cytoplasmic domain involved in migration and survival in primary oligodendrocyte precursors using cell-permeable homeopeptides.

The mapping of regions within integrin cytoplasmic domains responsible for the different effects on cell behaviour is an important part of an analysis of integrin-mediated signalling. In order to facilitate this analysis in primary cells, we have used cell-permeable homeopeptides to deliver sequences mimicking parts of the integrin beta1 cytoplasmic domain into the cell. In a study using oligodendrocyte precursors, the cells that give rise to myelin-forming oligodendrocytes during CNS development, we show that these peptides can be used to manipulate beta1 integrin signalling and that the regions of the cytoplasmic domain involved in migration and survival are distinct. Peptides mimicking the N-terminal portion of the cytoplasmic domain previously implicated in binding to Focal Adhesion Kinase (FAK) induce apoptosis, while peptides mimicking more C-terminal sequences do not cause cell death. In contrast they show that the NPIY sequence, the N-terminal one of two NPXY motifs previously implicated in signalling, is involved in migration. Peptides containing this sequence promote migration while alteration of NPIY to NPIA makes the peptide inhibitory to migration. Our results show that these peptides represent a novel approach to integrin signalling that allow rapid definition of critical cytoplasmic sequences in primary cells.

Amino Acid Sequence↗
PubMed1999

Postnatal booster injections increase engraftment after in utero stem cell transplantation.

BACKGROUND: The primary barrier to clinical application of in utero hematopoietic stem cell (HSC) transplantation is limited donor cell engraftment. We hypothesized that limited engraftment was due to competition between host and donor cells for available niches. We reasoned that increased engraftment might be achieved by performing multiple transplants separated by brief intervals to allow time for formation of new niches. To test this we performed multiple transplants in a congenic combination to avoid confounding immunologic effects. MATERIALS AND METHODS: C57Pep3B (H2Kb, CD45.1) mice were used as donors of adult bone marrow and C57Bl/6 (H2Kb, CD45.2) mice were used as 14-day-gestation fetal recipients. All fetuses were injected intraperitoneally with 1 x 10(6) mononuclear cells. Boosted neonates were injected at Days 2, 4, and 7 of life with 5 x 10(6) cells. All animals were analyzed for donor cell engraftment by dual-color flow cytometry using CD45 and CD45.1 antigens. Results are reported as the mean +/- SD. Statistical analysis was performed using the two-tailed Student t test with P < 0.05 considered significant. RESULTS: Postnatally boosted animals demonstrated significantly elevated levels of donor cell engraftment (3.30 +/- 0.8%; n = 8; P < 0.00001) when compared to the control animals (0.69 +/- 0.5%; n = 9) as determined by peripheral blood analysis at 6 weeks of age. This elevated level of engraftment was stable long term. CONCLUSIONS: Our results demonstrate a significant increase in donor cell engraftment with postnatal booster injections after in utero transplantation. This supports the hypothesis that a limited number of niches may be a major component of the barrier to engraftment. It also suggests that postnatal booster injections may be a viable therapeutic strategy for improving donor cell engraftment after in utero HSC transplantation.

Aging↗
PubMed1999

In utero bone marrow transplantation induces donor-specific tolerance by a combination of clonal deletion and clonal anergy.

BACKGROUND/PURPOSE: In utero bone marrow transplantation can induce donor-specific tolerance to postnatal solid organ transplantation, although the mechanisms remain poorly defined. In this study, we investigated the role of clonal deletion and clonal anergy in the maintenance of tolerance in a murine model of in utero bone marrow transplantation. METHODS: DBA/2 mice (MIs(a+)) were used as donors of adult bone marrow, and 14-day-gestation fetal Balb/c mice (MIs(a-)) were used as recipients. Tolerance was defined by donor-specific skin graft survival for more than 8 weeks. Clonal deletion was assessed by flow cytometry for Vbeta6 T cell receptor usage. A tolerant animal demonstrating partial deletion of CD4+/Vbeta6+ T cells and a nontolerant animal were selected for analysis of clonal anergy by a proliferation assay using plate-bound anti-Vbeta6 antibody for stimulation with or without exogenous interleukin-2 (IL2). RESULTS: Vbeta6+ splenocytes constituted 6.32% of CD4+ T cells in the tolerant animal compared with 9.19% in the nontolerant animal, demonstrating incomplete clonal deletion in the tolerant animal. Stimulation with plate-bound anti-Vbeta6 induced a good proliferative response in the nontolerant animal but a significantly attenuated response in the tolerant animal (P< .001), which was abrogated by the addition of IL2. CONCLUSIONS: In this murine model of in utero bone marrow transplantation, the tolerant state is characterized by partial clonal deletion of donor reactive T cells and clonal anergy of nondeleted donor reactive T cells. The anergic state can be abrogated by exogenous IL2, suggesting that the mechanism of anergy is a deficiency of IL2 production.

Animals↗
PubMed1999

Persistent postnatal transgene expression in both muscle and liver after fetal injection of recombinant adenovirus.

BACKGROUND/PURPOSE: Immune responses to both vector and transgene antigens have limited the efficacy of postnatal gene therapy. We hypothesize that the fetal period may offer immunologic and developmental advantages for successful gene therapy. In this study we examined the efficacy, persistence, and immunologic effects of recombinant adenovirus after intramuscular delivery into fetal mice. METHODS: E1-deleted adenovirus (AdCMVlacZ) containing the beta-galactosidase marker gene was used for injection. Fetal Balb/c mice (14 to 15 days' gestation) were injected with AdCMVlacZ in 10-microL volume in either the shoulder or hindlimb musculature. Animals were killed at 18 to 20 days' gestation and up to 4 months postnatally for analysis of transgene expression and adenoviral genome persistence. RESULTS: Fetuses were injected with doses of AdCMVlacZ from 1 x 10(8) to 2 x 10(10) viral particles (n = 80). Optimal survival rate was 83% at 18 to 20 days' gestation and 55% at 4 weeks of age using a dose of 1 x 10(9) particles. Expression of beta-galactosidasae at 18 to 20 days localized to multiple muscle groups surrounding the site of injection, as well as bone marrow stroma, liver, lung, and dorsal root ganglia. Persistent muscle and liver transgene expression was observed for as long as 16 and 8 weeks, respectively, after injection. The pattern of liver expression was confined to discrete foci of hepatocytes, which appeared to increase in size in older animals. No histological evidence of muscle or liver inflammation was observed at any time after injection. No neutralizing antibodies were observed postnatally. CONCLUSIONS: Our results confirm that gene therapy in the fetus may be advantageous. Distribution of vector in the fetus at the site of injection is clearly broader than in the adult setting. Furthermore, the absence of immune response and persistence of transgene expression suggests that fetal exposure to foreign transgene and vector antigens may induce tolerance. Although we have not proven genomic integration, the histological appearance of transgene expression in the liver supports this conclusion. By understanding the mechanisms that underlie persistent transgene expression, fetal gene therapy may become a feasible strategy for the treatment of fatal genetic diseases.

Adenoviridae↗
PubMed1999

Troubles with twins: fetoscopic therapy.

Monochorionic twin gestations present challenges in perinatal management not seen in dichorionic twins or singleton pregnancies. Monochorionic twin gestations may be complicated by (1) a monoamniotic presentation, (2) a structurally anomalous cotwin, (3) twin reversed arterial perfusion sequence, (4) and the twin-twin transfusion syndrome. Each of these conditions is associated with increased morbidity and mortality rates. Although a variety of therapeutic interventions are available, fetoscopic surgery is a promising new approach to these conditions. Advances in microinstrumentation have allowed for the wider application of fetoscopy for a variety of prenatally diagnosed conditions, including "troubles with twins." This article examines the natural history, pathophysiology, diagnosis, and indications for fetoscopic surgery in complicated monochorionic twin gestations.

Chorion↗
PubMed1999

Perinatal management of fetal malformations amenable to surgical correction.

Advances in prenatal imaging modalities such as ultrasound and magnetic resonance imaging have permitted the more frequent and accurate diagnosis of congenital malformations. Fetal surgical intervention has been reserved for those malformations that lead to fetal demise or are life-threatening in the neonatal period. We review the current indications for intervention in fetuses diagnosed with congenital lung lesions, congenital diaphragmatic hernia, sacrococcygeal teratoma, and obstructing airway lesions. A discussion of the recent interest in the fetal repair of severe myelomeningocele is also included.

Congenital Abnormalities↗
PubMed1999

An assessment of observer bias in the shunt design trial.

INTRODUCTION: An international multicenter randomized trial comparing standard pressure differential valves, Orbis Sigma valves and PS Medical Delta valves, for children with newly diagnosed hydrocephalus failed to show a difference in the time to first shunt failure (power 80%). In order to avoid observer bias, the primary endpoint, shunt failure, was defined by detailed clinical and radiological criteria. Surgeons notified the data center when their patient reached endpoint. Their decision was blindly reviewed by a centralized adjudication process. The current analysis asks: (1) Did the surgeons and the blinded adjudication process agree on the presence of shunt failure? (2) Do the shunt survival curves change when the surgeons' assessment of outcome is used? METHODS: The two methods of outcome assessment (surgeons' decision and adjudication process) were compared with a kappa statistic for the presence of shunt failure and a log rank statistic for time to shunt failure. RESULTS: The surgeon and the adjudication process agreed on the presence of the primary outcome in 313/344 patients in the trial (kappa = 0.81). The 31 cases of disagreement were evenly distributed among the three shunts. The survival curves generated from the surgeons' decision were not significantly different from those generated by the adjudication process (log rank = 0.70, p = 0.37) and did not alter the study conclusions. DISCUSSION: Observer bias in the assessment of outcome is always a concern in an unblinded clinical trial such as this one. We did not detect an observer bias of sufficient magnitude to alter the study conclusions.

Child↗
PubMed1999

The shunt design trial: variation in surgical experience did not influence shunt survival.

INTRODUCTION: An international multicenter randomized trial comparing standard pressure differential valves, Orbis Sigma valves and PS Medical Delta valves for children with newly diagnosed hydrocephalus failed to show a difference in the time to first shunt failure. Surgeons' prior experience with the three valves varied. This analysis was performed to assess whether lack of surgical experience with any of the valves could explain the overall negative result. METHODS: (1) Shunt survival was compared at high- and low-volume centers. (2) The 1-year shunt survival rates (+/-95% confidence interval) for patients entered in the first quarter and the last quarter of the 25-month accrual period were compared (for all patients, and for each shunt). RESULTS: Survival curves for high- and low-volume centers were similar. Ninety of the 344 randomized patients were accrued in the first quarter and 93 in the last quarter. The 1-year shunt survival for all patients entered in the first quarter was 72% (+/-11%) compared to 64% (+/-10%) for patients entered in the last quarter. The shunt-specific results were 66% (+/-20%) compared to 54% (+/-20%) for Delta valve patients, 75% (+/-20%) compared to 70% (+/-17%) for standard valve patients and 76% (+/-18%) compared to 66% (+/-16%) for Orbis Sigma patients. DISCUSSION: Shunt survival did not improve as surgeons accumulated experience over the course of the study. Although participating surgeons had varying levels of experience with the different shunts at the start of the trial, this does not appear to explain the overall negative trial result.

Adult↗
PubMed1999

Distinct roles for astrocyte alphavbeta5 and alphavbeta8 integrins in adhesion and migration.

The alphav integrins are likely to be an important group of molecules for regulating astrocyte behaviour within the central nervous system. Together with their ligand vitronectin, they are expressed by astrocytes in vivo and are further upregulated during neurological disease. Here we have characterised the expression of alphav integrins on primary astrocytes from both rat and mouse, and shown that they express just two members, alphavbeta5 and alphavbeta8. By using RGD peptides and function-blocking antibodies against the beta1 integrins and alphavbeta5, we find that both alphavbeta5 and alphavbeta8 can act as functional receptors for vitronectin. However, while alphavbeta5 is largely responsible for astrocyte adhesion to vitronectin this integrin appears to play no role in migration on vitronectin, with alphavbeta8 playing the dominant role in promoting migration on this substrate. beta1 integrins are not involved in mediating interactions between astrocytes and vitronectin. These results were confirmed in experiments with astrocytes derived from mice in which the beta5 gene had been deleted by homologous recombination. beta5 null astrocytes attached to vitronectin at a reduced rate, but showed no defect in migration on vitronectin relative to wild-type astrocytes. These data provide the first evidence that alphavbeta8 regulates migration and show that astrocyte alphavbeta5 and alphavbeta8 integrins have distinct functions.

Animals↗
PubMed1998

Comparison of in-vivo body composition using two Lunar dual-energy X-ray absorptiometers.

OBJECTIVE: To compare in-vivo composition analysis between two dual energy X-ray absorptiometers, a DPX and a DPX/L, from the same manufacturer (LUNAR), pre(Study A) and post(Study B) hardware changes on both absorptiometers. DESIGN: Comparison of (1) quality assurance (QA) data: air-counts low (38 keV), air-counts high (70 keV), air-counts ratio, percent spillover, R-delrin; and (2) total body compartments: total body tissue (TBTISS), total body fat (TBF), percent total body fat (%TBF), total body lean (TBLEAN), total body bone mineral content (TBBMC) and total body bone mineral density (TBBMD), between the two absorptiometers. SETTING: Centre for Bone and Body Composition Research, University of Leeds. SUBJECTS: Study A, 14 normal subjects and Study B, a different cohort of 19 normal subjects, were scanned on both machines on the same day. RESULTS: In Study A, large significant differences were observed in the QA parameters between the two machines. The DPX, air-counts low and air-counts high, being 25% and 22% lower than the DPX/L. The Bland Altman method of analysis indicated that the DPX was significantly higher for TBTISS (0.3 kg), %TBF (2%) and TBF ( 1.4 kg) and correspondingly lower for TBLEAN (-1.0 kg). No significant difference was observed in TBBMC. After the hardware changes (Study B) a marked reduction in the differences in QA air-counts was observed. The DPX air-counts low was now 1% higher and air-counts high 8% lower than the DPX/L. The DPX had now only small significant negative differences for %TBF (-0.6%) and TBF (-0.4 kg) and a small significant positive difference for TBLEAN (0.4 kg), compared to the DPX/L. TBBMC difference although slightly increased, was still non-significant. CONCLUSIONS: The closer agreement observed in the QA parameters after the hardware changes was associated with a reduction in the mean differences, 95%CI of the mean differences and limits of agreement of the comparison of body composition analysis from the Lunar machines using the Bland-Altman method. The study indicates that the QA limits set for bone mineral analysis may require more stringent limits for body composition.

Absorptiometry, Photon↗
PubMed1998

Randomized trial of cerebrospinal fluid shunt valve design in pediatric hydrocephalus.

OBJECTIVE: Forty percent of standard cerebrospinal fluid shunts implanted for the treatment of pediatric hydrocephalus fail within the first year. Two new shunt valves designed to limit excess flow, particularly in upright positions, were studied to compare treatment failure rates with those for standard differential-pressure valves. METHODS: Three hundred-forty-four hydrocephalic children (age, birth to 18 yr) undergoing their first cerebrospinal fluid shunt insertion were randomized at 12 North American or European pediatric neurosurgical centers. Patients received one of three valves, i.e., a standard differential-pressure valve; a Delta valve (Medtronic PS Medical, Goleta, CA), which contains a siphon-control component designed to reduce siphoning in upright positions; or an Orbis-Sigma valve (Cordis, Miami, FL), with a variable-resistance, flow-limiting component. Patients were monitored for a minimum of 1 year. Endpoints were defined as shunt failure resulting from shunt obstruction, overdrainage, loculations of the cerebral ventricles, or infection. Outcome events were assessed by blinded independent case review. RESULTS: One hundred-fifty patients reached an endpoint; shunt obstruction occurred in 108 (31.4%), overdrainage in 12 (3.5%), loculated ventricles in 2 (0.6%), and infection in 28 (8.1%). Sixty-one percent were shunt failure-free at 1 year and 47% at 2 years, with a median shunt failure-free duration of 656 days. There was no difference in shunt failure-free duration among the three valves (P = 0.24). CONCLUSION: Cerebrospinal fluid shunt failure, predominantly from shunt obstruction and infection, remains a persistent problem in pediatric hydrocephalus. Two new valve designs did not significantly affect shunt failure rates.

Adolescent↗
PubMed1998

Uptake of ethylenediamine tetramethylene phosphonic acid in normal bone after multiple applications. A non-human primate study.

Palliation of bone pain in patients with bone metastases has previously been evaluated using 153Sm (samarium) complexed to bone seeking ethylenediamine tetramethylene phosphonic acid (CAS 1429-50-1, EDTMP). Repeated application of the radioligand as needed was found progressively less effective. This study questions whether EDTMP exerts a blocking function, limiting access to bone or osseous tumours with successive administration. The pharmacokinetics and biodistribution of 153Sm-EDTMP in the normal experimental baboon (n = 6) during three successive applications (6 weekly) each with two different concentrations of EDTMP (0.7 and 1.4 mg/kg b.wt.) were investigated using bone scintigraphy. 153Sm-EDTMP (111 MBq) was injected in each case and monitored for 5 h. Curves of tracer kinetics and bone to background uptake were obtained, also blood and cumulative urine curves. Comparisons were statistically assessed in each group between successive applications and between EDTMP concentrations. Partial blocking with the low EDTMP concentration reached statistical significance after the third application. The first application of the high EDTMP concentration yielded lower uptake in the bone than did low EDTMP pointing to blocking by the high concentration, but not seen with repeated applications. Continual application of high concentration EDTMP could lead to a reduced level of calcium in serum and increased parathyroid hormone levels which might trigger osteoblastic activity and bone remodelling. This would partially affect the blocking which was thus more obvious at the low EDTMP concentration.

Animals↗
PubMed1997

Division of labor of Schwann cell integrins during migration on peripheral nerve extracellular matrix ligands.

Myelination of the peripheral nervous system (PNS) requires the migration of Schwann cells during both development and regeneration. We have characterized the expression pattern of Schwann cell integrins and analyzed their role in migration on different ECM substrates known to be present within the PNS. We found that Schwann cells in cell culture express four beta1 integrins, alpha1 beta1, alpha2 beta1, alpha6 beta1, and another unidentified beta1 integrin, as well as two alpha v integrins, alpha v beta3 and alpha v beta8. Using the Varani migration assay, we found that laminin-1, laminin-2 (merosin), and fibronectin increased Schwann cell migration, while vitronectin and collagen did not increase migration compared to an uncoated plastic substrate. Schwann cell migration on laminin-1 and laminin-2 (merosin) was blocked by antibodies against beta1 integrins, but not affected by RGD peptides or antibodies against beta3 integrins. In contrast, migration on fibronectin was unaffected by antibodies against beta1 and beta3 integrins but was blocked by RGD peptides. This in vitro study shows that there is a division of labor of Schwann cell integrins in the regulation of migration on peripheral nerve ECM components; beta1 integrins mediate migration on laminin-1 and laminin-2 (merosin), while alpha v integrins mediate migration on fibronectin. Taken together, these results suggest that multiple interactions between Schwann cell integrins and ECM within the PNS will contribute to Schwann cell migration during myelination of the PNS.

Animals↗
PubMed1997

Contrasting effects of mitogenic growth factors on oligodendrocyte precursor cell migration.

We have examined the effects of the mitogenic growth factors platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and glial growth factor-2 (GGF-2) on oligodendrocyte precursor migration. In an agarose drop migration assay PDGF and bFGF stimulated migration while GGF-2 had no effect. The migration-enhancing effect of bFGF cannot be blocked by neutralising antibodies against PDGF, confirming that this effect is direct and not mediated via upregulation of PDGF receptors. Based on our results, we propose a model in which the differing effects of PDGF and GGF-2 ensure appropriate numbers of oligodendrocyte precursor cells in the vicinity of axons to be myelinated during development.

Cell Division↗
PubMed1997

Understanding the molecular basis of cell migration; implications for clinical therapy in multiple sclerosis.

1. Multiple sclerosis is characterized by areas of demyelination spread throughout the central nervous system, in which the myelin sheaths surrounding axons are destroyed. While therapies aimed at suppressing the autoimmune response, such as beta-interferon, may prevent further damage, they cannot repair or replace the lost myelin. To this end, an additional therapy has been proposed, which involves transplanting cells of the oligodendrocyte lineage into the central nervous system. 2. The cell of interest for transplantation is the oligodendrocyte precursor because, unlike the differentiated cell, it is an intrinsically migratory and proliferative cell. In order to optimize the transplant strategy we have investigated the molecular mechanisms that control migration in vitro, so that these mechanisms might be upregulated to maximize cell migration in vivo. We have focused on the integrin family of cell adhesion molecules, known to play a fundamental role in the regulation of migration in other cell types. 3. These studies show that oligodendrocytes express a limited repertoire of integrins consisting of alpha 6 beta 1 and three different alpha v integrins. alpha 6 beta 1 is expressed throughout development but alpha v integrins show developmental regulation; differentiation is accompanied by loss of alpha v beta 1 and upregulation of alpha v beta 5. 4. Function-blocking studies show that oligodendrocyte precursor migration in vitro is mediated primarily by the developmentally regulated alpha v beta 1 integrin, but not alpha 6 beta 1 or alpha v beta 3. Taken together with previous evidence that cell migration can be regulated by altering integrin expression, this work suggests that modifying expression levels of alpha v beta 1 on oligodendrocyte precursors may increase the migratory capacity of these cells. If so, this would support a future therapeutic strategy aimed at transplanting genetically modified oligodendrocyte precursors to repair widespread demyelinated lesions.

Animals↗