Outcomes of haematopoietic stem cell transplantation for inherited metabolic disorders: a report from the Australian and New Zealand Children's Haematology Oncology Group and the Australasian Bone Marrow Transplant Recipient Registry.

We report a retrospective analysis of 53 haematopoietic stem cell transplants for inherited metabolic disorders performed at ANZCHOG transplant centres between 1992 and 2008. Indications for transplant included Hurler syndrome, ALD, and MLD. The majority of transplants utilized unrelated donor stem cells (66%) with 65% of those being unrelated cord blood. Conditioning therapy was largely myeloablative, with Bu plus another cytotoxic agent used in 89% of recipients. Primary graft failure was rare, occurring in three patients, all of whom remain long-term survivors following the second transplant. The CI of grade II-IV and grade III-IV acute GVHD at day +100 was 39% and 14%, respectively. Chronic GVHD occurred in 17% of recipients. TRM was 12% at day +100 and 19% at one yr post-transplant. OS at five yr was 78% for the cohort, 73% for patients with ALD and 83% for patients with Hurler syndrome. There was no statistically significant difference in overall survival between unrelated marrow and unrelated cord blood donor groups. The development of interstitial pneumonitis was an independent variable shown to significantly impact on TRM and OS. In summary, we report a large cohort of patients with inherited metabolic disorders with excellent survival post-allogeneic transplant.

Biochemical analysis of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of patients with Alzheimer's disease neuropathology.

Alzheimer's disease (AD) is characterized by selective vulnerability of specific neuronal populations within particular brain regions. For example, hippocampal glutamatergic cell populations within the CA1/subicular pyramidal cell fields have been found to be particularly vulnerable early in AD progression. In contrast, hippocampal GABA-ergic neurons and receptors appear resistant to neurodegeneration. Despite relative sparing of GABA(A) receptors in AD, it is possible that the specific subunit composition of these receptors may undergo alterations with disease progression. In order to address this issue, we employed quantitative Western blot analysis to examine protein levels of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of subjects displaying increasing severity of AD neuropathology. Subjects were categorized into three groups based upon Braak staging pathologic criteria: pathologically mild (stages I/II, n=9); moderate (stages III/IV, n=8); and severe (stages V/VI, n=7). Across all subject groups, levels of subunit protein were heterogeneously distributed throughout the five hippocampal subregions analyzed (subiculum, CA1-3, dentate gyrus). Statistical analyses revealed differential preservation of GABA(A) receptor subunits in AD. In particular, alpha 1, beta 1, and beta 2 displayed little difference in protein levels among pathologically mild, moderate, and severe subject groups. In contrast, although relatively modest, protein levels of the alpha 5 subunit were significantly reduced between subjects with severe neuropathology compared with pathologically mild subjects (13.5% reduction). Collectively, our data provide evidence for heterogeneous distribution and relative sparing of GABA(A) receptor subunits in the hippocampus of AD patients.

INK4A/ARF deletions are acquired at relapse in childhood acute lymphoblastic leukaemia: a paired study on 25 patients using real-time polymerase chain reaction.

Current risk-adjusted intensive therapies for childhood acute lymphoblastic leukaemia (ALL) are expected to result in an event-free survival of greater than 75%. In sharp contrast, relapsed paediatric ALL is a difficult disease to treat. In this study, 25 paediatric patients with ALL were analysed at diagnosis and relapse for their p16 (exon 2) status using the most accurate method of detection, real-time polymerase chain reaction (PCR). The median time to relapse for the group was 27 months. At diagnosis, the incidence of p16 homozygous and hemizygous deletion in this group was 32% and 20% respectively. The incidence of homozygous p16 deletion at relapse was 64%. A large number of patients, eight of 16 (50%), developed p16 homozygous deletion at relapse. Of those eight patients, four were hemizygous and four were germline at diagnosis. At diagnosis, those patients with a homozygous or hemizygous p16 deletion relapsed sooner than those germline for p16. We have shown that p16 alterations are frequently present in relapsed lymphoblastic leukaemia in children.

Hemizygous p16(INK4A) deletion in pediatric acute lymphoblastic leukemia predicts independent risk of relapse.

The genes at the INK4A/ARF locus at 9p21 are frequently involved in human cancer. Virtually all p16(INK4A) exon 2 (henceforth called p16) inactivation in pediatric acute lymphoblastic leukemia (ALL) occurs by gene deletion. The results of this study illustrate that real-time quantitative polymerase chain reaction is capable of detecting gene deletion in primary patient specimens with a precision not previously achieved by conventional methods. Importantly, this assay includes the detection of hemizygous deletions. The study revealed, strikingly, that the risk ratio for relapse for hemizygous deletion compared with no deletion was 6.558 (P =.00687) and for homozygous deletion was 11.558 (P =.000539). These results confirm and extend the authors' previous findings that homozygous deletion of p16 in pediatric ALL patients is an independent prognostic indicator of outcome from therapy.

Spinal section and opioid receptor blockade induce the appearance of Fos-like immunoreactivity in the spinal cord of the decerebrated rabbit.

The expression of Fos-like immunoreactivity has been studied in spinal segments L5-S1 of decerebrated, unanaesthetized, but otherwise unstimulated rabbits. The aim of the study was to establish baseline levels of Fos in such preparations, and to examine how these might change after spinalization and opioid receptor blockade. In animals with an intact spinal cord, approximately 30 Fos-positive profiles per section were found in the superficial dorsal horns (i.e. laminae I and II) of each 40-microm section, while about 20 profiles per section were found immediately adjacent to the central canal (lamina X). Fos-like immunoreactive profiles were rare elsewhere in the gray matter. When the spinal cord was sectioned at L1 (after blockade with local anaesthetic), significantly more Fos-like immunoreactivity was found in superficial and central regions of the gray matter (approximately 90 profiles per section) in animals perfused 4 h after decerebration, but not when perfusion was performed 2 or 8 h after decerebration. The opioid antagonist naloxone (0.25 mg/kg/h) had little effect on expression of Fos-like immunoreactivity in spinalized preparations, but significantly increased the numbers of Fos-positive profiles in all but the ventral areas of the spinal gray matter in non-spinalized preparations. The present data show that spinal section induces a transient increase in expression of Fos in the superficial and central parts of the spinal gray matter. It appears that spinalization induces spontaneous activity in some neurons in these regions of the cord, presumably as a result of relief of descending inhibition. The effects of naloxone indicate that endogenous opioids exert tonic inhibition over Fos-expressing spinal neurons in non-spinalized rabbits.

The prevalence of hepatitis C in survivors of paediatric malignancy.

OBJECTIVE: To assess the prevalence of hepatitis C in 200 patients with paediatric malignancies, surviving in remission more than 5 years from diagnosis, who had received blood product transfusions before 1990 when routine screening of blood products for hepatitis C began. METHOD: The second and third generation Abbott Diagnostics ELISA was used to assess hepatitis C seropositivity. Seropositive patients and those with abnormal liver transaminases were assessed by hepatitis C virus RNA polymerase chain reaction (PCR). RESULTS: A low incidence (4%) of seropositivity for hepatitis C was found in survivors of paediatric malignancy who were transfused prior to routine screening of blood products in this cohort. CONCLUSIONS: All patients identified have evidence of hepatitis and may be at high risk of developing cirrhosis.

Tetralogy of Fallot repair results in activation of the renin-angiotensin-aldosterone system.

Fluid retention is a common problem following transannular patch repair of tetralogy of Fallot. The present study was undertaken to evaluate whether humoral substances may contribute to this process. Patients undergoing tetralogy of Fallot repair using a transannular patch technique were compared to patients undergoing simple ventricular septal defect repair. Hormone levels were determined by radioimmunoassay. Fluid retention was defined as pleural effusions persisting beyond 5 days. The data demonstrate that patients undergoing tetralogy of Fallot repair had elevated levels of anti-diuretic hormone, renin, angiotensin II, and aldosterone as compared to a group with ventricular septal defect. The elevation in hormone levels in the tetralogy of Fallot group was principally accounted for by those who developed effusions postoperatively. These results suggest that activation of the renin angiotensin-aldosterone system may be one of the contributing factors to fluid retention following tetralogy of Fallot repair.

Bedside optometric care in a hospital environment.

The demand for bedside optometric care is increasing because of the rise in the geriatric population and the increasing fiscal demands with which long-term care facilities in the United States are faced. It is important for optometrists to keep pace with the need for bedside optometric care in hospital environments. I will discuss the requirements for performing the bedside examination such as knowing what rules and regulations must be followed, the importance of making an environmental assessment with corresponding recommendations, as well as possessing the necessary portable equipment.

Low vision: physician screening helps to improve patient function.

Low vision is a common problem of older patients. As a primary care practitioner, you are not expected to treat most ocular problems, but you can play an important role by identifying patients with visual impairment. Familiarize yourself with the normal visual changes that occur with aging as well as the more common age-related ocular diseases, including glaucoma, cataracts, age-related macular degeneration, and diabetic retinopathy. You can identify patients with visual problems with a careful history and basic in-office tests of visual acuity. Refer patients found to have visual impairment to an eye care specialist for further evaluation.

Renin, angiotensin II, and the development of effusions following bidirectional Glenn and Fontan procedures.

Pleural effusions are a troublesome complication following bidirectional Glenn and Fontan procedures. It was our hypothesis that effusions may be related to alterations in hormones that regulate fluid homeostasis. We made serial determinations (by radioimmunoassay) of antidiuretic hormone, cortisol, aldosterone, angiotensin II, and renin in patients undergoing bidirectional Glenn (n = 16) and Fontan procedures (n = 24). There were six patients who developed effusions following surgery. These patients had a different endocrinological pattern characterized by persistent elevation in renin (28 +/- 9 vs 9 +/- 5 ng/mL per hour, p < 0.01) and angiotensin II (110 +/- 33 vs 33 +/- 14 ng/L, p < 0.01) on the fifth postoperative day as compared to patients who did not develop effusions. These data demonstrate that patients who develop effusions following bidirectional Glenn and Fontan procedures have activation of their renin-angiotensin system.

Damus-Kaye-Stansel procedure: midterm follow-up and technical considerations.

The Damus-Kaye-Stansel operation is useful in the management of complex congenital heart defects. We reviewed our experience with 23 patients who underwent a Damus-Kaye-Stansel procedure. The anastomotic technique was individualized depending on the anatomy. The aortic and pulmonary artery incisions were carried into the sinuses of Valsalva in 9 patients, the aorta was transected in 11 patients, and a patch was used to augment the anastomosis in 13 patients. Concurrent procedures included a Fontan operation (n = 9, mortality = 0), right ventricle-pulmonary artery conduit (n = 5, mortality = 0), bidirectional Glenn procedure (n = 6, mortality = 1), and central aortopulmonary shunt (n = 3, mortality = 2; emergency = 1). Survival is 87% with a median follow-up of 7 years (range, 2 months to 9.2 years). Four patients underwent late revision of the Damus-Kaye-Stansel connection. All survivors are asymptomatic. We conclude that the Damus-Kaye-Stansel connection provides excellent midterm results when the proximal anastomosis is adapted to the anatomy of the patient.

Age-related vision changes: a primary care guide.

Visual impairment becomes more prevalent with age. A person has "low vision" when legally blind or partially sighted. In addition, many older patients have functional visual impairment that affects their ability to read, even with corrective lenses. Normal visual changes that occur with aging include presbyopia, decreased contrast sensitivity, decreased dark/light adaptation, and delayed glare recovery. The four most prevalent age-related ocular diseases are macular degeneration, open-angle glaucoma, cataract, and diabetic retinopathy. The primary care practitioner can identify patients with visual problems with a careful history and in-office tests of visual acuity. Patients with visual impairments should be referred to an eye care practitioner for a more comprehensive ocular evaluation.

Reduction in triiodothyronine levels following modified Fontan procedure.

Diminished cardiac function is a common manifestation following the modified Fontan procedure. Since thyroid hormone has important effects on cardiovascular function, the present study was undertaken to evaluate changes in thyroid hormone levels following this operation. A control group consisting of children undergoing open heart procedures other than a Fontan procedure was also evaluated. Serum total and free triiodothyronine (T3), total and free thyroxine (T4), thyroid stimulating hormone (TSH), and thyroglobulin were measured by immunoassays. The Fontan group demonstrated an initial increase in free T4, while free T3, total T3, total T4, TSH, and thyroglobulin were reduced. Over the subsequent days, free T4 decreased to below the preoperative value. By the fifth and eighth postoperative days, free T3, total T3, free T4, and total T4 remained reduced, while TSH and thyroglobulin began increasing toward the preoperative levels. The control group also demonstrated decreases in free T3 and TSH. However, these values had returned to baseline by the fifth postoperative day. The results indicate that children undergoing open heart surgery have suppression of the pituitary-thyroid axis, and that this is prolonged in patients undergoing Fontan procedure. The decreased levels of T3 following Fontan procedure may have adverse effects on the recovery of patients undergoing this operation.

Management of mycotic aneurysm following repair of corrected transposition.

Omental flaps have been used for the treatment of infected aortic prosthetic grafts in adults. We report the case of a 26-month-old male who developed a mycotic aneurysm 9 months following insertion of a ventricle to pulmonary artery conduit. The aneurysm was excised and the reconstructed outflow tract wrapped with omentum. The patient has not had recurrence of his aneurysm following this course of management.

Selective tolerance to discriminative stimulus effects of morphine or d-amphetamine.

Pharmacological selectivity of tolerance to the discriminative stimulus effects of morphine or d amphetamine was examined in pigeons trained to discriminate among 3.2mg/kg morphine, saline, and 1.8mg/kg d-amphetamine under a 3-key fixed-ratio 30 schedule of food delivery. Cumulative doses of morphine (0.32-10mg/kg) or d-amphetamine (0.10-3.2mg/kg) evoked morphine-or d-amphetamine-key responding, respectively, in a dose-dependent manner. Suspending training and administering repeated doses of morphine (32mg/kg b.i.d.) for 1 week increased the dose of morphine required for morphine-key responding approximately 5-fold, without altering sensitivity to d-amphetamine. Conversely, repeated treatment with d-amphetamine (5.6mg/kg b.i.d.) increased the dose of d-amphetamine required for d-amphetamine-key responding approximately 7-fold, without decreasing sensitivity to morphine. Repeated treatment with saline (1ml/kg b.i.d.) for 1 or 2 weeks did not alter sensitivity to stimulus effects of either morphine or d-amphetamine. Sensitivity to stimulus effects of morphine recovered fully within 1 week after morphine treatment ended; sensitivity to stimulus effects of d-amphetamine recovered partially within 3 days after d-amphetamine treatment ended. For morphine, but not for d-amphetamine, increases in the dose required for stimulus effects were accompanied by increases in the dose required for rate-reducing effects. These results demonstrate that tolerance to discriminative stimulus effects of morphine and d-amphetamine is pharmacologically selective and suggest that pharmacotherapies targeted to one drug of abuse may produce little change in sensitivity to subjective effects of drugs from a different pharmacological class.

Physical and chemical considerations in the in vitro calibration of microdialysis probes for biogenic amine neurotransmitters and metabolites.

The object of the present study was to examine the effects of temperature, oxidation, and pH on in vitro relative recovery of catecholamine and indoleamine neurotransmitters and their metabolites using microdialysis probes. Relative recovery of norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), homovanillic acid (HVA), and 5-hydroxytryptamine (5HT) increased with temperature from 0 to 46 degrees C. For each compound, the increase in the amount recovered with increasing temperature was different. The stability of norepinephrine and dopamine was not affected at any temperature using deoxygenated calibration standard solutions containing ascorbic acid but was greatly reduced when exposed to ambient air without antioxidant treatment; catecholamine metabolites and the indole compounds were less affected. No change for in vitro relative recovery was observed by varying the pH of the perfusing solution from 6 to 8. Thus, temperature control in probe calibration as well as analyte stability using antioxidant treatment are important in reducing the error when estimating extracellular concentrations of neurotransmitter and metabolites.

Thrombus-free, human endothelial surface in the midregion of a Dacron vascular graft in the splanchnic venous circuit--observations after nine months of implantation.

The addition of an endothelial cell lining to a prosthetic vascular graft may reduce the thrombogenicity of the blood-contacting surface. An endothelialized mesoatrial graft was implanted in a patient with Budd-Chiari syndrome caused by a primary inferior vena caval leiomyosarcoma. During the initial surgery a Dacron vascular graft was preclotted with plasma and then lined with microvascular endothelial cells derived from the patient's subcutaneous adipose tissue. The patient did well initially but 9 months later required resection of a mechanical stricture of the graft that occurred as it passed beneath the costochondral junction. Grossly, the luminal surface of the resected graft was free of thrombus, with a smooth, glistening, white surface. Light microscopy demonstrated a surface layer of cells morphologically consistent with an endothelial cell monolayer, a subendothelial layer composed of extracellular matrix and spindle-shaped cells, and granulation tissue around the Dacron fabric. Immunohistochemistry and electron microscopy confirmed the presence of vascular endothelium on the luminal surface. This report documents the successful achievement of a human endothelial cell monolayer that persisted for 9 months in the midportion of a Dacron vascular graft.

Human microvessel endothelial cell isolation and vascular graft sodding in the operating room.

We have evaluated multiple factors inherent to an operating room-compatible endothelial cell procurement and sodding procedure. Microvessel endothelial cell isolations have been performed on fat tissue obtained from over 140 patients with a 100% success rate. Liposuction-derived fat was optimal with respect to cell yield, and isolation time. The devices and equipment used were acceptable to the operating room and the complete cell procurement procedure was successful even in the hands of personnel with minimal training. Fat digestion was achieved using crude clostridial collagenase, with an average cell yield of 1 x 10(6) microvessel endothelial cells/gm of fat. Evaluation of this procedure with canine fat using an operating room acceptable procedure resulted in a 100% procurement success rate requiring 1.5 hours (+/- .5 hrs) for completion of the fat isolation, and cell isolation procedure. Microvessel EC could subsequently be used in graft seeding or sodding techniques to establish endothelial cell monolayers on vascular grafts. Our results indicate that one person with minimal cell isolation background can reproducibly isolate large quantities of sterile autologous endothelial cells in the operating room for immediate use in endothelial cell seeding/sodding procedures.

Comparison of sonicated and nonsonicated specimens for the isolation of Chlamydia trachomatis.

Cultures of sonicated and nonsonicated chlamydial specimens were compared using a Heat Systems sonicator with a cup-horn attachment for sonication. Chlamydial inclusions were detected in 71 of 780 cultures tested; 44 (62%) were positive from both, while 12 (16.9%) were positive in nonsonicated only, and 15 (21.1%) in sonicated specimens only. The mean number of inclusions from sonicated specimens was more than double that from nonsonicated specimens. The cultures of sonicated specimens could be screened more quickly because of statistically significant higher inclusion counts, less toxicity, and the absence of overlying epithelial cells.