Growth and Nutritional Outcomes in Children Post-Haematopoietic Stem Cell Transplant without Exposure to Total Body Irradiation.

AIMS: Poor growth in childhood cancer survivors who undergo haematopoietic stem cell transplant (HSCT) without exposure to radiation is reported anecdotally, although literature to support this is limited. The aims of this study were to assess the change in height standard deviation score (SDS) and the final adult height (FAH) in children who underwent chemotherapy-only conditioned HSCT and to identify predictors of poor growth. MATERIALS AND METHODS: We conducted a retrospective hospital medical record review (1984-2010) of children (1-10 years) who underwent chemotherapy-only conditioned HSCT, noting anthropology measurements at cancer diagnosis, HSCT, 10 years old and FAH. RESULTS: The median age at HSCT of the 53 patients was 4.5 years, 75% had a haematological malignancy and 25% a solid tumour. Half of the cohort underwent allogenic HSCT and most (89%) conditioned with busulphan. The mean change in height SDS from primary cancer diagnosis to FAH was -1.21 (±1.18 SD), equivalent to 7-8.5 cm loss, with a mean FAH of -0.91 SDS (±1.10 SD). The greatest height loss occurred between diagnosis and HSCT (-0.77 SDS, 95% confidence interval -1.42, -0.12, P = 0.01), with no catch-up growth seen by FAH. Patients with solid tumours had the greatest height loss. Overall body mass index SDS did not change significantly over time, or by cancer type. CONCLUSIONS: Chemotherapy-only conditioned HSCT during childhood can impact FAH, with the greatest height loss occurring prior to HSCT and no catch-up growth after treatment finishes. Children transplanted for a solid tumour malignancy seem to be more at risk, possibly due to intensive treatment regimens, both pre-transplant and during conditioning.

Adenovirus-mediated delivery of catalase to retinal pigment epithelial cells protects neighboring photoreceptors from photo-oxidative stress.

Oxidative stress is involved in the pathogenesis of many diseases. Overexpression of antioxidant enzymes by gene therapy may protect tissues from oxidative damage. Because the reactive oxygen species hydrogen peroxide can diffuse across cell membranes, we hypothesized that overexpression of the antioxidant catalase within certain cells might protect neighboring cells. To test this hypothesis, we transduced retinal pigment epithelial (RPE) cells in vitro and in vivo with adenovirus carrying the catalase gene (Ad.CMV.catalase). After transduction of only a subset of RPE cells in vitro, all cells in the culture were protected from exogenous hydrogen peroxide. Similarly, in vivo, eyes injected with Ad. CMV. catalase had high catalase levels in the RPE, which protected the adjacent photoreceptors from light damage and reduced photoreceptor oxidative stress as measured by the markers 4-hydroxynonenal and nitrotyrosine. Both in vitro and in vivo, gene therapy with Ad. CMV. catalase protected neighboring cells from oxidative stress-induced terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity. The data provide a paradigm for antioxidant gene therapy with catalase, designed to protect not only transduced cells, but also neighboring cells.

Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy.

Recombinant vectors based on adeno-associated virus (AAV) can efficiently transduce many different cell types, including cells of the retina, resulting in stable gene expression. A major shortcoming of this vector is its small packaging capacity. A trans-splicing approach, which reconstitutes gene expression from two independent AAV vectors, can be used to overcome the vector's packaging limitations. The efficiency of this system to date has been disappointing, and therefore its utility for therapeutic application limited. We demonstrate here that efficiency and cellular specificity of trans-splicing is dependent on selection of the appropriate AAV serotype. Efficiency of transgene expression resulting from trans-splicing in skeletal muscle approaches that obtained when delivering the intact transgene when using AAV2 vectors packaged with AAV5 capsids (AAV2/5). This expands the potential of AAV vectors for retinal gene therapy. The use of AAV2/5 also increases the efficiency of trans-splicing in photoreceptors. Selection of the appropriate AAV serotype is likely to affect efficiency of trans-splicing in other organ systems as well.

Comparison of external jugular and central venous pressures in mechanically ventilated patient.

We compared central venous pressures, measured via a 150 mm triple lumen catheter in the internal jugular vein with simultaneous external jugular venous pressures, measured with a 5 mm cannula in the external jugular vein, in 24 patients undergoing major surgery. Patients were mechanically ventilated in the supine position. Six sets of paired measurements of mean central venous pressure and mean external jugular venous pressure were taken by a blinded observer, in random order and at end-expiration at 30-min intervals during surgery. Four patients were not studied because of a failure to cannulate the external jugular vein. The remaining 20 patients yielded 111 sets of paired measurements. The mean difference between external jugular venous pressure and central venous pressure was 0.3 mmHg over a range of central venous pressure of 0-22 mmHg. Limits of agreement were 3.6 to +3.0 mmHg (95% CI 4.1 to +3.5 mmHg). We conclude that external jugular venous pressure is an accurate estimate of central venous pressure in surgical patients undergoing mechanical ventilation.

Effects of remifentanil and alfentanil on the cardiovascular responses to induction of anaesthesia and tracheal intubation in the elderly.

BACKGROUND: We compared the effects of remifentanil and alfentanil on arterial pressure and heart rate at induction of anaesthesia and tracheal intubation in 40 ASA I-III patients aged greater than 65 yr, in a randomized double-blind study. METHODS: Patients received either remifentanil 0.5 microg kg(-1) over 30 s, followed by an infusion of 0.1 microg kg min(-1) (group R) or alfentanil 10 microg kg(-1) over 30 s, followed by an infusion of saline (group A). Anaesthesia was then induced with propofol, rocuronium, and 1% isoflurane with 66% nitrous oxide in oxygen. RESULTS: Systolic arterial pressure (SAP) and mean arterial pressure (MAP) decreased after the induction of anaesthesia (P<0.05) and increased for 3 min after intubation in both groups (P<0.05), but remained below baseline values throughout. Heart rate remained stable after induction of anaesthesia but increased significantly from baseline after intubation for 1 and 4 min in groups R and A, respectively (P<0.05). There were no significant between-group differences in SAP, MAP, and heart rate. Diastolic pressure was significantly higher in group A than group R at 4 and 5 min after intubation (P<0.05). Hypotension (SAP < 100 mm Hg) occurred in four patients in group R and three patients in group A. CONCLUSIONS: Remifentanil and alfentanil similarly attenuate the pressor response to laryngoscopy and intubation, but the incidence of hypotension confirms that both drugs should be used with caution in elderly patients.

Allogeneic bone marrow transplant improves outcome for juvenile myelomonocytic leukaemia.

OBJECTIVE: To correlate clinical presentation and therapeutic outcomes in children with a diagnosis of juvenile myelomonocytic leukaemia. METHODS: The medical records of 14 children who fulfilled the International Juvenile Myelomonocytic Leukaemia Working Group Criteria for a diagnosis of juvenile myelomonocytic leukaemia (JMML) presenting to a single institution were reviewed, and their clinical status at September 2000 was documented. RESULTS: The most common presenting features were hepatosplenomegaly and lymphadenopathy. Fifty per cent of cases presented in the first year of life. Nine of 14 patients initially received chemotherapy otherwise used in the treatment of acute myeloid or lymphoblastic leukaemia with no apparent benefit. All six patients who received conditioning therapy with chemotherapy alone, followed by allogeneic bone marrow transplant (BMT), are in complete remission at a median follow-up duration of 12 months (range 5-91 months). Five of six patients surviving post-allogeneic BMT received marrow from an unrelated donor. Only one of seven patients who did not receive BMT survived long-term. CONCLUSION: Children with a diagnosis of JMML should be treated with allogeneic BMT as soon as a suitable donor is found. The role of anti-leukaemic therapy in this disease, prior to BMT, requires further investigation in the context of a multicentre clinical trial.

Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.

Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3-4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2-4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.

Long-term protection of retinal structure but not function using RAAV.CNTF in animal models of retinitis pigmentosa.

The present study aimed to determine whether intravitreal administration of an adeno-associated virus (AAV) carrying ciliary neurotrophic factor (CNTF) can achieve long-term morphological and physiological rescue of photoreceptors in animal models of retinitis pigmentosa, and whether injection of this virus after degeneration begins is effective in protecting the remaining photoreceptors. We injected rAAV.CNTF.GFP intravitreally in early postnatal Prph2(Rd2/Rd2) (formerly rds/rds) mice and in adult P23H and S334ter rhodopsin transgenic rats. Contralateral eyes received an intravitreal injection of rAAV.GFP or a sham injection. We evaluated the eyes at 6 months (rats) and 8.5 to 9 months (mice) postinfection and looked for histological and electoretinographic (ERG) evidence of photoreceptor rescue and CNTF-GFP expression. Intravitreal administration of rAAV resulted in efficient transduction of retinal ganglion cells in the Prph2(Rd2/Rd2) retina, and ganglion, Muller, and horizontal/amacrine cells in the mutant rat retinas. Transgene expression localized to the retinal region closest to the injection site. We observed prominent morphological protection of photoreceptors in the eyes of all animals receiving rAAV.CNTF.GFP. We found the greatest protection in regions most distant from the CNTF-GFP-expressing cells. The Prph2(Rd2/Rd2) ERGs did not exhibit interocular differences. Eyes of the rat models administered rAAV.CNTF.GFP had lower ERG amplitudes than those receiving rAAV.GFP. The discordance of functional and structural results, especially in the rat models, points to the need for a greater understanding of the mechanism of action of CNTF before human application can be considered.

Comparison of effects of remifentanil and alfentanil on cardiovascular response to tracheal intubation in hypertensive patients.

In a randomized double-blind study, we compared the effect of remifentanil and alfentanil on the cardiovascular response to laryngoscopy and tracheal intubation in patients on long-term treatment for hypertension. Forty ASA II-III patients were allocated to receive (i) remifentanil 0.5 microg kg(-1) followed by an infusion of 0.1 microg kg min(-1) or (ii) alfentanil 10 microg kg(-1) followed by an infusion of saline; all patients received glycopyrrolate 200 microg before the study drug. Anaesthesia was induced with propofol and rocuronium and maintained with 1% isoflurane and 66% nitrous oxide in oxygen. Laryngoscopy and tracheal intubation were performed after establishment of neuromuscular block. Arterial pressure and heart rate (HR) were measured non-invasively at 1 min intervals from 3 min before induction until 5 min after intubation. Systolic (SAP), diastolic and mean arterial pressure decreased significantly after induction in both groups (P<0.05). Maximum increases in mean SAP after laryngoscopy and intubation were 35 and 41 mm Hg in the remifentanil and alfentanil groups, respectively. After intubation, arterial pressure did not increase above baseline values in either group. HR remained stable after induction of anaesthesia, but increased above baseline values after intubation. Mean maximum HR was 87 beats min(-1) for the remifentanil group (12 beats min(-1) above baseline; P=0.065) and 89 beats min(-1) for the alfentanil group (15 beats min(-1) above baseline; P<0.05). There were no significant differences between groups in HR or arterial pressure at any time. There were no incidences of bradycardia. Seven patients in the remifentanil group and four in the alfentanil group received ephedrine for hypotension (i.e. SAP<100 mm Hg).

Strain-dependent anterior segment neovascularization following intravitreal gene transfer of basic fibroblast growth factor (bFGF).

BACKGROUND: A promising strategy for delaying death of photoreceptor cells in retinal degenerative disease is to support survival of these cells through intraocular delivery of growth/neurotrophic factors. One factor that has received great attention is basic fibroblast growth factor (bFGF; fgf-2), a known stimulator of angiogenesis. We evaluated the potential for neovascularization induced by adenovirus-mediated intravitreal delivery of bFGF. METHODS: Recombinant adenoviruses carrying the low molecular weight (18 kD) or the high molecular weight (22, 23 and 24 kD) forms of human bFGF, driven by the cytomegalovirus (CMV) promoter/enhancer, were prepared. Viruses were delivered to eyes of different strains of mice and rats through intravitreal injection. Contralateral eyes were injected with control virus carrying a reporter gene [green fluorescent protein (GFP) or lacZ]. Transgene expression was assessed by Western analysis and by immunohistochemistry. Neovascularization was evaluated in vivo and histologically at termination of the experiment. RESULTS: Adenovirus-mediated delivery of the 18 kD form of bFGF resulted in anterior segment neovascularization in a strain-dependent fashion. Generation of new blood vessels was not observed after injection of the higher molecular weight forms of bFGF or of control solutions. CONCLUSION: The low molecular weight form (18 kD) (but not the high molecular weight forms) of bFGF drives angiogenic response in the anterior segment of specific strains of mice. Genetic modifiers may contribute to and/or prevent neovascularization induced by bFGF.

Pharmacologically regulated gene expression in the retina following transduction with viral vectors.

The availability of inducible expression systems makes regulatable control of therapeutic proteins an attainable goal in gene therapy. We delivered tetracycline-inducible transgenes to the subretinal space using recombinant adenoviruses. Upon administration of doxycycline, we demonstrated reversible expression of green fluorescent protein in the retinal pigment epithelium as well as modulation of human growth hormone produced in the retina and secreted in the blood stream. This mode of delivery and regulation offers a unique way to evaluate gene function in the eye and represents a novel method for introducing therapeutic proteins into the retina.

Gene therapy restores vision in a canine model of childhood blindness.

The relationship between the neurosensory photoreceptors and the adjacent retinal pigment epithelium (RPE) controls not only normal retinal function, but also the pathogenesis of hereditary retinal degenerations. The molecular bases for both primary photoreceptor and RPE diseases that cause blindness have been identified. Gene therapy has been used successfully to slow degeneration in rodent models of primary photoreceptor diseases, but efficacy of gene therapy directed at photoreceptors and RPE in a large-animal model of human disease has not been reported. Here we study one of the most clinically severe retinal degenerations, Leber congenital amaurosis (LCA). LCA causes near total blindness in infancy and can result from mutations in RPE65 (LCA, type II; MIM 180069 and 204100). A naturally occurring animal model, the RPE65-/- dog, suffers from early and severe visual impairment similar to that seen in human LCA. We used a recombinant adeno-associated virus (AAV) carrying wild-type RPE65 (AAV-RPE65) to test the efficacy of gene therapy in this model. Our results indicate that visual function was restored in this large animal model of childhood blindness.

AAV-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse.

Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. The present study aimed to test whether adeno-associated virus (AAV)-mediated delivery of the gene encoding CNTF delays photoreceptor death in the rhodopsin knockout (opsin(-/-)) mouse, an animal model of RP. The vector was made to express a secretable form of CNTF in tandem with a marker GFP. Cultured 293 cells transduced with this virus expressed both CNTF and GFP. The conditioned media from such cells supported the survival of chick dorsal root ganglion neurons in the same manner as recombinant CNTF. Subretinal administration of this virus led to efficient transduction of photoreceptors as indicated by GFP fluorescence and CNTF immunostaining. Histologic examination showed significant photoreceptor preservation in the injected quadrant of the retina. This protection lasted through termination of the experiment (3 months). AAV-mediated delivery of CNTF may have implications for the treatment of human retinal degeneration.

Intraocular delivery of recombinant virus.

In the past 5 yr, advances in technology have been made that allow efficient somatic transfer of functional genes to target cells in the eye in vivo. The ability to deliver functional genes to these cells is due primarily to the development of viral vectors-viruses in which various replicative functions have been replaced with transgene cassettes. Because progress continues in developing new vectors and refining those that are already available, the field has moved past the step of actually proving that gene transfer is possible to one where vectors are used (experimentally) for therapeutic purposes. In fact, proofs of principle of virus-based retinal gene therapy have been achieved in relevant animal models for retinitis pigmentosa (1-3).

Generation of transgenic mice for studies of ocular development and disease.

Over the past decade, there has been spectacular growth in our understanding of the molecular genetics of eye development and ocular disease. Although this is primarily caused by developments in recombinant DNA technology, it is also caused in large part by advances in, and the spread of, transgenic mouse technology. Whereas 10 years ago few laboratories had the equipment and skill to generate transgenic mice, now most investigators have access to a transgenic core facility. Transgenic mouse studies have fueled our understanding of ocular development, have delineated regulatory elements involved in gene expression in cells of the eye, and have unraveled pathogenic mechanisms involved in eye disease.

Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa.

PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.