Biomedical subjects

L Lawrence

Publications and source records attributed to L Lawrence.

At least 19 recordsLinked to original sources

PubMed2006

Clinically applicable procedure for gene delivery to fetal gut by ultrasound-guided gastric injection: toward prenatal prevention of early-onset intestinal diseases.

Targeting gene therapy vectors to the fetal intestinal tract could provide a novel means toward prevention of the early postnatal intestinal pathology of cystic fibrosis and other conditions, such as congenital enteropathy, that cause intestinal failure. Among these conditions, cystic fibrosis is by far the most common lethal genetic disease. It is caused by a functional absence or deficiency of the cystic fibrosis transmembrane conductance regulator and manifests in the gut as meconium ileus. Prenatal treatment of genetic disease may avoid early-onset tissue damage and immune sensitization, and may target cells that are less accessible in the adult. We investigated gene transfer to the fetal gut, using a minimally invasive injection technique. First-generation replication-deficient adenoviral vectors encoding the beta-galactosidase gene and transduction-enhancing agents were injected into the stomach of early-gestation fetal sheep (n = 8, 60 days of gestation; term, 145 days) under ultrasound guidance. Reporter gene expression was observed 2 days after injection in the villi of the gastrointestinal epithelia after 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining and beta-galactosidase immunohistochemistry of fetal tissues. Expression of beta-galactosidase, as measured by enzyme-linked immunosorbent assay, was enhanced after pretreatment of the fetal gut with sodium caprate, which opens tight junctions, and after adenovirus complexation with DEAE-dextran, which confers a positive charge to the virus. Instillation of the fluorocarbon perflubron after virus delivery resulted in tissue transduction from the fetal stomach to the colon. Using a clinically relevant technique, we have demonstrated widespread gene transfer to the fetal gastrointestinal epithelia.

Adenoviridae↗

PubMed2005

Factors influencing adenovirus-mediated airway transduction in fetal mice.

Intra-amniotic injection of adenovirus allows transduction of the fetal airways following natural fetal breathing movements. This administration method is promising for use in gene therapy for cystic fibrosis and other diseases for which the main target for exogenous gene expression is the lung. Here we have investigated factors that may affect the efficacy of gene transfer to the murine fetal lung. We examined marker compound distribution and transgene expression (from a first-generation adenoviral vector) at different stages of development. This demonstrated that fetal breathing movements at 15-16 days of gestation are of sufficient intensity to carry marker/vector into the fetal lungs. These movements can be significantly stimulated by the combination of intra-amniotic theophylline administration and postoperative exposure of the dam to elevated CO(2) levels. However, the most important factor for efficient and consistent pulmonary transgene delivery is the dose of adenoviral vector used, as both the degree of transduction and the percentage of lungs transduced increases with escalating viral dose.

Adenosine↗

PubMed2004

Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero.

Cystic fibrosis is a common lethal genetic disease caused by functional absence of the cystic fibrosis transmembrane conductance regulator (CFTR). Although a candidate disease for in utero gene therapy, demonstration of potentially therapeutic levels of transgene expression in the fetal airways after minimally invasive gene delivery is a mandatory prerequisite before application of this approach in humans can be considered. We report here on the delivery of a beta-galactosidase expressing adenovirus directly to the airways of fetal sheep in utero using ultrasound-guided percutaneous injection of the trachea in the fetal chest. Injection of adenoviral particles to the fetal airways was not associated with mortality and resulted in low-level expression in the peripheral airways. However, complexation of the virus with DEAE dextran, which confers a positive charge to the virus, and pretreatment of the airways with Na-caprate, which opens tight junctions, increased transgene expression, and a combination of these two enhancers resulted in widespread and efficient gene transfer of the fetal trachea and bronchial tree. Using a percutaneous ultrasound-guided injection technique, we have clearly demonstrated proof of principle for substantial transgene delivery to the fetal airways providing levels of gene expression that could be relevant for a therapeutic application of CFTR expressing vectors.

Adenoviridae↗

PubMed2004

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy.

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.

Animals↗

PubMed2003

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.

Animals↗

PubMed2001

A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex.

Vascular endothelial growth factor (VEGF) is produced by cancer cells in response to hypoxia and is the primary stimulant of vascularization in solid tumors. Endothelial cells lining the blood vessels of these tumors have a high concentration of receptor-bound VEGF on their surface, providing a target for antibody- directed cancer therapy. To obtain a cloned antibody to this target when bound to its receptor on tumor endothelium, we used phage display technology to create a single-chain Fv (sFv) antibody library from mice immunized with the 165-amino acid isoform of human VEGF-A. We selected, purified, and characterized LL4, an anti-VEGF sFv that was shown to react with receptor-bound VEGF. LL4 bound selectively to blood vessel endothelium, as shown by immunohistochemistry on tissue sections of human tumors. Furthermore, using autoradiography and grain counting of histological sections, systemically administered LL4 was shown to localize selectively to the endothelial lining of tumor blood vessels in human colorectal carcinoma xenografts in vivo. This study demonstrates the feasibility of targeting tumor vasculature using recombinant antibodies to the VEGF:receptor complex.

Animals↗

PubMed2001

Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis.

We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.

Animals↗

PubMed2001

Phenotype of the endothelium in the human term placenta.

The placental endothelium contributes to regulating transplacental exchange and maintaining the immunological maternofetal barrier. We characterized the endothelial phenotype in human normal term placentae with a panel of antibodies to endothelial antigens using a standardized immunofluorescence method. Placental endothelium strongly expressed vWF, PAL-E, H-antigen, thrombomodulin, PECAM-1, CD34, CD36, ICAM-1, CD44, thy-1, A10/33-1, VE-cadherin, caveolin-1 and HLA-G, whereas occludin, claudin-1, eNOS, angiotensin converting enzyme (ACE), ICAM-2, endoglin and integrin-alphathetabeta(3)were weakly expressed. PGI(2)synthase, tissue factor, E-selectin and VCAM-1 were not detected. Some antigens were heterogenously expressed along the vascular tree or within individual villi. Expression of ACE, eNOS, vWF, P-selectin, E-selectin, integrin alpha(v)beta(3)and endoglin was stronger in the maternal decidual vessels, while PECAM-1, CD44, thy-1 and caveolin-1 expression was stronger in fetal vessels. Some endothelial markers were present in trophoblasts and stroma. Endothelial proliferation was apparent in mature intermediate and terminal villi. There was limited inflammatory response to TNFalpha in explants, characterized by upregulation of vWF, P-selectin, PECAM-1 and CD44, downregulation of thrombomodulin, but no increase in ICAM-1 expression, nor induction of E-selectin, VCAM-1 or tissue factor. These patterns of heterogeneity, proliferative activity and inflammatory activation may underlie the specific physiological roles of the placental endothelium.

Adult↗

PubMed1999

6-oxa isosteres of anacardic acids as potent inhibitors of bacterial histidine protein kinase (HPK)-mediated two-component regulatory systems.

A series of 6-oxa isosteres of anacardic acids (6-higher alkyl/alkenyl-2-hydroxybenzoic acids) was synthesised and several members were discovered to be among the most potent inhibitors (IC50 values < or = 5 microM) of the bacterial two-component regulatory systems, KinA/SpoOF and NRII/NRI, reported to date. The Gram-positive antibacterial activity in selected strains is also presented.

Anacardic Acids↗

PubMed1999

1997 summary: National Hospital Discharge Survey.

OBJECTIVES: This report presents national estimates of the use of non-Federal short-stay hospitals in the United States during 1997. Numbers and rates of discharges, diagnoses, and procedures are shown by age and sex. Discharges are also shown by geographic region of the hospital. Average lengths of stay are presented for all discharges and for selected diagnostic categories by age and by sex. METHODS: The estimates are based on medical abstract data collected through the National Hospital Discharge Survey for 1997. The survey has been conducted annually by the National Center for Health Statistics since 1965. Diagnoses and procedures presented are coded according to the International Classification of Diseases, 9th Revision, Clinical Modification, or ICD-9-CM.

Adolescent↗

PubMed1999

National hospital discharge survey: annual summary, 1997.

OBJECTIVES: This report presents national estimates of the use of non-Federal short-stay hospitals in the United States during 1997 and selected trend data. Estimates are provided by demographic characteristics of patients discharged, geographic region of hospitals, conditions diagnosed, and surgical and nonsurgical procedures performed. Measurements of hospital use include number and rate of discharges and days of care, and the average length of stay. METHODS: The estimates are based on data collected through the National Hospital Discharge Survey. The survey has been conducted annually by the National Center for Health Statistics since 1965. In 1997, data were collected for approximately 300,000 discharges. Of the 501 eligible non-Federal short-stay hospitals in the sample, 474 (95 percent) responded to the survey. Diagnoses and procedures were coded according to the International Classification of Diseases, 9th Revision, Clinical Modification, or ICD-9-CM.

Adolescent↗

PubMed1999

Detailed diagnoses and procedures, National Hospital Discharge Survey, 1997.

OBJECTIVES: This report presents national estimates of the use of non-Federal short-stay hospitals in the United States during 1997. Estimates of first-listed diagnoses, all-listed diagnoses, days of care for first-listed diagnoses, and all-listed procedures are shown by sex and age of patient and geographic region of hospital. METHODS: The estimates are based on data collected through the National Hospital Discharge Survey for 1997. The survey has been conducted annually by the National Center for Health Statistics since 1965. In 1997 data were collected for approximately 300,000 discharges from 474 non-Federal short-stay hospitals. Diagnoses and procedures are presented according to their code number in the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM).

Adolescent↗

PubMed1998

Ambulatory surgery in the United States, 1996.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures performed on an ambulatory basis in hospitals and freestanding ambulatory surgery centers in the United States during 1996. Data are presented by types of facilities, age and sex of the patient, and geographic region. Major categories of procedures and diagnoses are shown by age, sex, and region. METHODS: The estimates are based on data collected by the 1996 National Survey of Ambulatory Surgery conducted by the National Center for Health Statistics. The 1996 data were abstracted from 125,000 medical records in 488 hospitals and freestanding ambulatory surgery centers.

Adolescent↗

PubMed1998

Substrate, inhibitor, or antibody stabilizes the Glu 119 Gly mutant influenza virus neuraminidase.

We have previously reported the isolation and characterization of an influenza virus variant with decreased sensitivity to the neuraminidase-specific inhibitor zanamivir. This variant, which has a mutation in the active site, Glu 119 Gly (E119G), has the same specific activity as the wild-type neuraminidase (NA), but is inherently unstable, as measured by loss of both enzyme activity and NC10 monoclonal antibody reactivity. However, despite the instability of the NA, replication of the virus in liquid culture is not adversely affected. We demonstrate here that in addition to enhanced temperature sensitivity the mutant NA was significantly more sensitive to formaldehyde and to specimen preparation for electron microscopy. Substrate, inhibitor, or monoclonal antibodies stabilized the NA against all methods of denaturation. These results suggest that the instability of the variant is primarily at the level of polypeptide chain folding rather than at the level of association of monomers into tetramers. Furthermore the presence of high levels of substrate, either cell or virus associated, may be sufficient to stabilize the NA during virus replication.

Animals↗

PubMed1998

Structure of chordae tendineae in the left ventricle of the human heart.

The bicuspid (mitral) valve complex of the human heart consists of functional units which include the valve leaflets, chordae tendineae and the papillary muscles. The mechanical properties of these functional units depend to a large extent on the link between the muscle and the valve. This link is usually arranged in a branching network of avascular tendinous chordae composed of collagen and elastic fibres, which transmit contractions of the papillary muscle to the valve leaflets. In order to perform their function efficiently, the chordae have to possess a high degree of elasticity, as well as considerable strength and endurance. Human chordae tendineae originating from the left ventricles were obtained from 7 embalmed cadavers and 6 postmortem subjects of various ages. Samples washed in saline were fixed or postfixed in 9 % formol saline. Observations were made by illuminating the chordae along their axes. The reflected images originating from the superficial collagenous layers of the relaxed chordae showed a striped pattern 11 microm in width. Scanning electron and light microscopy of the chordae confirmed an undulating pattern of collagen fibrils arranged in bundles of planar waves in register and around the entire circumference of the chorda. The dimensions of the waves correlated with those of the striped reflected pattern. The observed undulating arrangement of the collagen fibrils appears to produce an inherent built-in elasticity which is likely to be of considerable advantage for a tissue which is under continuous repetitive stress. The chordae were covered by endocardium composed of a superficial layer of smooth squamous endothelial cells and an underlying dense layer of elastic fibres. It is suggested that the relaxed striped chordae, consisting of undulating collagen fibrils, straighten when the chordae become stretched by papillary muscle contraction, thereby mitigating the peak stress developed during muscle contraction. On relaxation the elastic tissue tends to return the collagen to its wavy configuration. It is also suggested that the regular wavy pattern of collagen seen in young individuals gradually changes with age by elongation of the wave pattern which eventually becomes randomised. In addition, with increasing age, substantial cushions of connective tissue appear below endocardium while the dense collagenous core has a reduced cross-sectional area which may lead to stretching and eventual rupture of the chordae.

Adolescent↗

PubMed1997

Ambulatory surgery in the United States, 1995.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures performed on an ambulatory basis in hospitals and freestanding ambulatory surgery centers in the United States during 1995. Data are presented by types of facilities, age, sex, and geographic region. Major categories of procedures and diagnoses are shown by age, sex, and region. METHODS: The estimates are based on data collected from the 1995 National Survey of Ambulatory Surgery conducted by the National Center for Health Statistics. The 1995 data were abstracted from 122,000 medical records in 489 hospitals and freestanding ambulatory surgery centers.

Adolescent↗

PubMed1997

Ambulatory surgery in the United States, 1994.

OBJECTIVES: This report presents estimates of surgical and nonsurgical procedures done on an ambulatory basis in hospitals and freestanding ambulatory surgery centers in the United States during 1994. Data are presented by types of facilities, age, sex, geographic region, and disposition for ambulatory surgery visits. Major categories of procedures and diagnoses are shown by age, sex, and region. METHODS: The estimates are based on data collected from the national Survey of Ambulatory Surgery, conducted for the first time in 1994 by the national Center for Health Statistics. The 1994 data were abstracted from 118,000 medical records in 494 hospitals and freestanding ambulatory surgery centers.

Adolescent↗

PubMed1997

Differential distribution of an endothelial barrier antigen between the pial and cortical microvessels of the rat.

An endothelial barrier antigen (EBA), reported to be a marker for endothelial cells (EC) displaying blood-brain barrier (BBB) characteristics, was probed with a monoclonal antibody in pial and cortical microvessels in rat brain. In contrast to the uniform labelling of EC in cortical vessels, pial microvessels showed a heterogeneity in EBA expression. Most pial vessels consisted of a mixture of EBA positive and EBA negative cells whereas a smaller number of vessels were either completely negative or uniformly positive. Significantly, in vessels showing incomplete expression it was typically EC furthest from the brain surface that did not express EBA. Although the function of EBA is unknown, the variable expression in pial microvascular EC may be related to their incomplete barrier characteristics.

Animals↗